U.S. patent application number 12/856066 was filed with the patent office on 2011-02-17 for methods, compositions, and kits for generating rrna-depleted samples or isolating rrna from samples.
This patent application is currently assigned to EPICENTRE TECHNOLOGIES CORPORATION. Invention is credited to Roy R. Sooknanan.
Application Number | 20110040081 12/856066 |
Document ID | / |
Family ID | 43586862 |
Filed Date | 2011-02-17 |
United States Patent
Application |
20110040081 |
Kind Code |
A1 |
Sooknanan; Roy R. |
February 17, 2011 |
METHODS, COMPOSITIONS, AND KITS FOR GENERATING rRNA-DEPLETED
SAMPLES OR ISOLATING rRNA FROM SAMPLES
Abstract
The present invention provides methods, compositions, and kits
for generating rRNA-depleted samples and for isolating rRNA from
samples. In particular, the present invention provides compositions
comprising affinity-tagged antisense rRNA molecules corresponding
to substantially all of at least one rRNA molecule (e.g., 28S, 26S,
25S, 18S, 5.8S and 5S eukaryotic cytoplasmic rRNA molecules, 12S
and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S and
5S prokaryotic rRNA molecules) and methods for using such
compositions to generate rRNA-depleted samples or to isolate rRNA
molecules from samples.
Inventors: |
Sooknanan; Roy R.;
(Beaconsfield, CA) |
Correspondence
Address: |
Casimir Jones, S.C.
2275 DEMING WAY, SUITE 310
MIDDLETON
WI
53562
US
|
Assignee: |
EPICENTRE TECHNOLOGIES
CORPORATION
Madison
WI
|
Family ID: |
43586862 |
Appl. No.: |
12/856066 |
Filed: |
August 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61234044 |
Aug 14, 2009 |
|
|
|
Current U.S.
Class: |
536/24.31 ;
536/23.1 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12N 15/1006 20130101; C12Q 1/6806 20130101; C12Q 2563/131
20130101; C12Q 2525/117 20130101 |
Class at
Publication: |
536/24.31 ;
536/23.1 |
International
Class: |
C07H 21/02 20060101
C07H021/02 |
Claims
1. A method of generating a rRNA-depleted sample from an initial
sample comprising: a) providing; i) an initial sample comprising
RNA molecules derived from at least one eukaryotic organism or
species, wherein said RNA molecules comprise rRNA molecules and
non-rRNA RNA molecules; ii) a composition comprising antisense rRNA
molecules complementary to substantially all of the sequence of at
least one rRNA molecule selected from the group consisting of: 25S,
26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules
and 12S and 16S eukaryotic mitochondrial rRNA molecules, wherein
said antisense rRNA molecules comprise affinity-tags at a ratio of
at least one affinity-tag per every 10 nucleobases of said
antisense rRNA molecules; and iii) a binding matrix comprising
affinity-tag-binding molecules; b) contacting said initial sample
with said composition comprising antisense rRNA molecules under
conditions such that at least some of said affinity-tagged
antisense rRNA molecules and at least some of said rRNA molecules
form double-stranded rRNA hybrids, thereby generating a treated
sample; c) contacting said treated sample with said binding matrix
under conditions wherein the ratio of said affinity-tag-binding
molecules to said affinity tags present in the antisense rRNA
molecules used in step b) is at least 8 to 1, such that at least a
portion of said double-stranded rRNA hybrids bind to said binding
matrix and are removed from said treated sample, thereby generating
an rRNA-depleted sample, wherein said rRNA-depleted sample
comprises at least a portion of said non-rRNA RNA molecules present
in said initial sample and is substantially depleted or free of
rRNA molecules that exhibit sequences exhibited by said at least
one rRNA molecule.
2. The method of claim 1, wherein said at least one eukaryotic
organism or species is selected from the group consisting of a
human, an animal, a plant, and a fungal organism or species.
3. The method of claim 2, wherein said RNA molecules in said
initial sample and said at least one rRNA molecule are derived from
the same eukaryotic organism or species.
4. The method of claim 1, wherein said RNA molecules are from a
first eukaryotic organism or species, and wherein said at least one
rRNA molecule is from a second eukaryotic organism or species.
5. The method of claim 4, wherein one of said first or second
organisms or species is Homo sapiens, and wherein the other of said
first or second organisms or species is a non-human mammal.
6. The method of claim 1, wherein said affinity-tags are associated
with at least one nucleobase selected from the group consisting of:
adenine (A), cytosine (C), guanine (G) and uracil (U); and wherein
said affinity-tags are present on said antisense rRNA molecules at
a ratio of at least one affinity-tag per every 8 nucleobases of
said antisense rRNA molecules; and/or wherein the ratio of said
affinity-tag-binding molecules to said affinity tags during said
contacting in step b) is at least 10 to 1.
7. The method of claim 1, wherein the number of molecules that
exhibit the sequence of said at least one rRNA molecule in said
rRNA-depleted sample is depleted by 99.0% relative to the number of
molecules that exhibit the sequence of said at least one rRNA
molecule in said initial sample.
8. The method of claim 1, wherein said at least one rRNA molecule
includes either both said 28S rRNA molecules and said 18S rRNA
molecules, or both said 25S or 26S rRNA molecules and said 16S rRNA
molecules.
9. The method of claim 1, wherein said at least one rRNA molecule
additionally includes both said 5.8 S rRNA and said 5S rRNA
molecules, and/or wherein said at least one rRNA molecule
additionally includes both said 12S and said 16S eukaryotic
mitochondrial rRNA molecules, and/or wherein said at least one rRNA
molecule is additionally selected from the group consisting of 16S,
23S, 4.5S and 5S eukaryotic chloroplast rRNA molecules.
10. The method of claim 1, wherein said initial sample comprising
RNA molecules derived from a eukaryotic organism or species
additionally comprises RNA molecules derived from at least one
prokaryotic species, wherein said prokaryotic RNA molecules
comprise rRNA molecules and non-rRNA RNA molecules, and wherein
said method further comprises: providing a composition that
additionally comprises affinity-tagged antisense rRNA molecules
complementary to substantially all of the sequence of at least one
rRNA molecule selected from the group consisting of 23S, 16S, and
5S prokaryotic rRNA molecules; and contacting said initial sample
with said composition that additionally comprises prokaryotic
affinity-tagged antisense rRNA molecules under conditions such that
at least some of said affinity-tagged antisense rRNA molecules and
at least some of said prokaryotic rRNA molecules form
double-stranded rRNA hybrids thereby generating a treated sample;
and contacting said treated sample with said binding matrix in step
c) under conditions wherein said rRNA-depleted sample also
comprises at least a portion of said non-rRNA prokaryotic RNA
molecules present in said initial sample and is substantially
depleted or free of prokaryotic rRNA molecules that exhibit
sequences exhibited by said at least one prokaryotic rRNA
molecule.
11. The method of claim 1, wherein at least a portion of said RNA
molecules in said initial sample are fragmented.
12. The method of claim 1, wherein said RNA molecules in said
initial sample comprise RNA extracted, isolated, or purified from a
source selected from the group consisting of: a tissue sample, a
cell sample, a paraffin-embedded sample, a paraffin-embedded
formalin-fixed (FFPE) sample, and an environmental sample
consisting of soil, water, growth medium, or a biological fluid or
specimen.
13. The method of claims 1, wherein said conditions in step b)
comprise incubating in the presence of a hybridization buffer at a
temperate of about 60-75.degree. C. for a first time period and
incubating at about room temperature for a second time period, and
wherein said binding matrix in step c) comprises a plurality of
individual particles or a binding column, and wherein said
conditions in step c) include at least occasional mixing at room
temperature and/or at 35-60.degree. C.
14. The method of claims 1, wherein the affinity-tag comprises
biotin.
15. A composition comprising antisense rRNA molecules complementary
to substantially all of the sequence exhibited by at least one rRNA
molecule selected from the group consisting of: 25S, 26S, 28S, 18S,
5.8S, and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, 16S, 23S, 4.5S and 5S
eukaryotic chloroplast rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules, wherein said antisense rRNA molecules
comprise affinity-tags at a ratio of at least one affinity-tag per
every 10 nucleobases of said antisense rRNA molecules, or wherein
said antisense rRNA molecules comprise affinity-tags at a ratio of
at least one affinity-tag per every 8 nucleobases of said antisense
rRNA molecules.
16. The composition of claim 15, wherein said antisense rRNA
molecules comprise multiple different anti-sense rRNA molecules
complementary to rRNA molecules selected from the groups consisting
of: at least four rRNA molecules selected from 25S, 26S, 28S, 18S,
5.8S, and 5S eukaryotic cytoplasmic rRNA molecules; two rRNA
molecules consisting of 12S and 16S eukaryotic mitochondrial rRNA
molecules; four rRNA molecules consisting of 16S, 23S, 4.5S and 5S
eukaryotic chloroplast rRNA molecules; and three rRNA molecules
consisting of 23S, 16S, and 5S prokaryotic rRNA molecules.
17. The composition of claim 15, additionally comprising RNA
molecules derived from at least one organism or species, wherein
said RNA molecules comprise rRNA molecules and non-rRNA RNA
molecules, wherein at least some of said affinity-tagged antisense
rRNA molecules in said composition are present as double-stranded
hybrids with at least some of said rRNA molecules.
18. The composition of claim 17, wherein the said RNA molecules
comprise RNA molecules derived from multiple organisms or
species.
19. The composition of claim 17, further comprising a binding
matrix comprising affinity-tag-binding molecules, wherein the ratio
of said affinity-tag-binding molecules to said affinity tags
present in the antisense rRNA molecules used in step b) is at least
8 to 1, or wherein the ratio of said affinity-tag-binding molecules
to said affinity tags present in the antisense rRNA molecules used
in step b) is at least 10 to 1.
20. A composition comprising an rRNA-depleted sample comprising
non-rRNA RNA molecules, wherein, compared to a sample that is not
rRNA-depleted, said composition is substantially free of rRNA
molecules that exhibit the sequence of at least one rRNA molecule
selected from the group consisting of: 25S, 26S, 28S, 18S, 5.8S,
and 5S eukaryotic cytoplasmic rRNA molecules; 12S and 16S
eukaryotic mitochondrial rRNA molecules; 16S, 23S, 4.5S and 5S
eukaryotic chloroplast rRNA molecules; and 23S, 16S, and 5S
prokaryotic rRNA molecules.
Description
[0001] The present application claims priority to U.S. Provisional
Application Ser. No. 61/234,044, filed Aug. 14, 2009, which is
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods, compositions, and
kits for generating rRNA-depleted samples and for isolating rRNA
from samples. In particular, the present invention provides
compositions comprising affinity-tagged antisense rRNA molecules
that exhibit sequences complementary to substantially all of at
least one full-length rRNA molecule encoded by a rRNA gene (e.g.,
compositions comprising affinity-tagged antisense rRNA molecules
that exhibit sequences which, either alone or in combination, are
complementary to substantially all or the complete sequence of at
least one rRNA molecule selected from among 28S, 26S, 25S, 18S,
5.8S and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, and 23S, 16S and 5S
prokaryotic rRNA molecules) and methods for using such compositions
to generate rRNA-depleted samples or to isolate rRNA from
samples.
BACKGROUND OF THE INVENTION
[0003] Since rRNA comprises about 95% to about 98% of the RNA in a
cell, its presence can complicate various types of analyses of
other RNA molecules of interest in a sample (e.g., gene expression
analyses by arrays or microarrays, next-generation sequencing of
tagged cDNA molecules made from one or more types of RNA molecules
in samples (e.g., using the massively parallel digital sequencing
methods referred to as "RNA-seq"), etc.). The problems caused by
rRNA are especially difficult for analyses of RNA molecules of
interest that are fragmented. For example, a considerable and
continuing problem in the art is to find better methods for
removing degraded rRNA from formalin-fixed paraffin-embedded (FFPE)
tissue sections. If better methods were available to remove
degraded rRNA from samples (e.g., FFPE-derived samples), it is
believed that the enormous quantities of clinical specimens, for
which medical outcomes of various diseases and various treatments
are recorded in the medical records, would provide extremely
valuable information related to identifying RNAs involved in the
cause, maintenance, response, diagnosis, or prognosis of many
diseases, such as cancer. Still further, better methods for
removing rRNA, including degraded rRNA, from non-rRNA RNA molecules
of interest would greatly improve the applicability and success of
methods that comprise deliberately degrading the RNA as part of the
particular method (such as the method of Ingolia et al., Science
324: 218-23, 2009, herein incorporated by reference).
SUMMARY OF THE INVENTION
[0004] The present invention provides methods, compositions, and
kits for generating rRNA-depleted samples or for isolating rRNA
from samples. In particular, the present invention provides methods
for generating compositions comprising affinity-tagged antisense
rRNA molecules, kits comprising such compositions, and methods for
using such compositions to generate rRNA-depleted samples or to
isolate rRNA from a sample (e.g., for further analysis and
use).
[0005] In some embodiments, the present invention provides methods
for generating a composition comprising affinity-tagged antisense
rRNA molecules that exhibit sequences which, either alone or in
combination, are complementary to the complete sequence of at least
one rRNA molecule selected from among 28S, 26S, 25S, 18S, 5.8S and
5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic
mitochondrial rRNA molecules, and 23S, 16S and 5S prokaryotic rRNA
molecules, wherein the composition is for use in a method for
generating a rRNA-depleted sample or for isolating rRNA from a
sample. In some embodiments, the composition comprises
affinity-tagged antisense rRNA molecules that exhibit sequences
which, either alone or in combination, are complementary to
multiple different rRNA molecule selected from among 28S, 26S, 25S,
18S, 5.8S and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, and 23S, 16S and 5S
prokaryotic rRNA molecules from one or multiple cells or organisms.
In some embodiments, the present invention provides methods for
using the composition comprising the affinity-tagged antisense rRNA
molecules for generating a rRNA-depleted sample and/or for
isolating rRNA from a sample.
[0006] In some embodiments, the present invention provides a
composition comprising affinity-tagged antisense rRNA molecules
that, either alone or in combination, exhibit one or more sequences
that are complementary to substantially all of the sequence
exhibited by at least one full-length rRNA molecule selected from
among 28S, 26S, 25S, 18S, 5.8S and/or 5S eukaryotic cytoplasmic
rRNA molecules, and 12S and 16S eukaryotic mitochondrial rRNA
molecules. In some embodiments, the present invention provides a
composition comprising affinity-tagged antisense rRNA molecules
that, either alone or in combination, exhibit one or more sequences
that are complementary to substantially all of the sequence
exhibited by the at least one full-length rRNA molecule selected
from among 23S, 16S and 5S prokaryotic rRNA molecules. In
particular embodiments, the at least one full-length cytoplasmic
rRNA molecule includes both the 28S rRNA and the 18S rRNA (or both
the 25S rRNA and the 18S rRNA, or both the 26S rRNA and the 18S
rRNA) from one or multiple eukaryotic cells, tissues, organs, or
organisms. In some embodiments, the at least one full-length rRNA
molecule includes both the 23S rRNA and the 16S rRNA from one or
multiple prokaryotic organisms. In some embodiments wherein the at
least one full-length rRNA molecule includes both the 28S rRNA and
the 18S rRNA (or both the 25S rRNA and the 18S rRNA, or both the
26S rRNA and the 18S rRNA) from one or multiple eukaryotic cells,
tissues, organs, or organisms, the affinity-tagged antisense rRNA
molecules also correspond to the 5.8S rRNA molecule and the 5S rRNA
molecule from the one or multiple eukaryotic cells, tissues,
organs, or organisms. In some embodiments, the affinity-tagged
antisense rRNA molecules also correspond to at least one
full-length rRNA molecule that includes both the 12S and 16S
eukaryotic mitochondrial rRNA molecules from the one or multiple
eukaryotic cells, tissues, organs, or organisms. In some
embodiments, the affinity-tagged antisense rRNA molecules also
correspond to at least one full-length rRNA molecule that includes
both the 23S rRNA and the 16S rRNA from one or multiple prokaryotic
organisms, and in some embodiments, the affinity-tagged antisense
rRNA molecules also correspond to at least one full-length rRNA
molecule that includes the 5S rRNA molecules from the one or
multiple prokaryotic organisms. In further embodiments of any of
the compositions comprising affinity-tagged antisense rRNA
molecules, the compositions are substantially free of non-rRNA RNA
molecules comprising the affinity tags. In further embodiments of
any of the compositions comprising affinity-tagged antisense rRNA
molecules, the compositions further comprise a binding matrix
comprising affinity-tag-binding molecules.
[0007] In some embodiments wherein the composition comprising
affinity-tagged antisense rRNA molecules corresponds to all of at
least one rRNA molecule selected from among 28S, 26S, 25S, and 18S
eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic
mitochondrial rRNA molecules, and prokaryotic 23S and 16S rRNA
molecules, said affinity-tagged antisense rRNA molecules are
fragmented (e.g., by controlled nuclease fragmentation or by
controlled fragmentation using a divalent metal cation, such as
Mg2+, and heat). In some embodiments, the composition comprising
fragmented affinity-tagged antisense rRNA molecules comprises
fragments ranging in size from about 3,500 nucleotides to about 240
nucleotides.
[0008] In some embodiments, the present invention provides a method
for generating a composition comprising affinity-tagged antisense
rRNA molecules comprising: a) generating double-stranded DNA
molecules comprising an RNA polymerase promoter that directs RNA
synthesis of antisense RNA corresponding to all of at least one
rRNA molecule selected from among 25S, 26S, 28S, 18S, 5.8S, and 5S
eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic
mitochondrial rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA
molecules; and b) contacting the double-stranded DNA molecules
comprising the RNA polymerase promoter with an RNA polymerase and
ribonucleoside-5'-triphosphates complementary to all of the
nucleobases, including at least one pair of
ribonucleoside-5'-triphosphates complementary to the same
nucleobase, one of which pair comprises an affinity tag and the
other of which pair does not comprise an affinity tag, and
incubating under conditions wherein affinity-tagged antisense rRNA
molecules are generated corresponding to substantially all of the
sequence of the at least one rRNA molecule.
[0009] With respect to such methods, work conduct during the
development of embodiments of the present invention determined that
the concentration of the ribonucleoside-5'-triphosphate molecules
comprising the affinity tag relative to the concentration of the
ribonucleoside-5'-triphosphate molecules that lacked the affinity
tag in the at least one pair of ribonucleoside-5'-triphosphates
complementary to the same nucleobase was important for generating a
composition comprising affinity-tagged antisense rRNA molecules
that could be used to remove >95% of the at least one rRNA
molecules from a sample, and that the relative concentration of the
ribonucleoside-5'-triphosphate molecules comprising the affinity
tag needed to be higher than had been previously used in the art.
In some embodiments of the present method for generating a
composition comprising antisense rRNA molecules, at least about 35%
of the ribonucleoside-5'-triphosphate molecules comprising the at
least one pair of ribonucleoside-5'-triphosphates comprise an
affinity tag and about 65% of the ribonucleoside-5'-triphosphate
molecules comprising the pair do not have an affinity tag. In some
embodiments of this method, at least about 40% of the
ribonucleoside-5'-triphosphate molecules comprising the at least
one pair of ribonucleoside-5'-triphosphates have an affinity tag
and about 60% of the ribonucleoside-5'-triphosphate molecules
comprising the pair do not have an affinity tag. In some
embodiments of this method, at least about 50% of the
ribonucleoside-5'-triphosphate molecules comprising the at least
one pair of ribonucleoside-5'-triphosphates have an affinity tag
and about 50% of the ribonucleoside-5'-triphosphate molecules
comprising the pair do not have an affinity tag. In some
embodiments of this method, at least about 60% of the
ribonucleoside-5'-triphosphate molecules comprising the at least
one pair of ribonucleoside-5'-triphosphates have an affinity tag
and about 40% of the ribonucleoside-5'-triphosphate molecules
comprising the pair do not have an affinity tag. In some
embodiments of this method, wherein the at least one rRNA molecule
is selected from 5.8S and 5S eukaryotic cytoplasmic rRNA, and
prokaryotic 5S rRNA, at least about 75% of the
ribonucleoside-5'-triphosphate molecules comprising the at least
one pair of ribonucleoside-5'-triphosphates have an affinity tag
and 25% of the ribonucleoside-5'-triphosphate molecules comprising
the pair do not have an affinity tag.
[0010] In some other embodiments, the present invention provides a
method for generating a composition comprising affinity-tagged
antisense rRNA molecules comprising: a) generating double-stranded
DNA molecules comprising an RNA polymerase promoter that directs
RNA synthesis of antisense RNA corresponding to all of at least one
rRNA molecule selected from among 25S, 26S, 28S, 18S, 5.8S, and 5S
eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic
mitochondrial rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA
molecules; b) contacting the double-stranded DNA molecules
comprising the RNA polymerase promoter with an RNA polymerase and
ribonucleoside-5'-triphosphates complementary to all of the
nucleobases, including at least one ribonucleoside-5'-triphosphate
that comprises an affinity-tag-reactive moiety (e.g., an allylamino
group on the 5 position of uridine), and incubating under
conditions wherein antisense rRNA molecules comprising the
affinity-tag-reactive moiety are generated, which antisense rRNA
molecules correspond to substantially all of the sequence of the at
least one rRNA molecules; and c) contacting the antisense rRNA
molecules comprising the affinity-tag-reactive moiety with a
quantity of an affinity tag reagent (e.g., Biotin-X-X-NHS,
EPICENTRE Biotechnologies, Madison, Wis.) under conditions wherein
the affinity tag reagent reacts with at least a portion of the
affinity-tag-reactive moieties in the antisense rRNA molecules
comprising the affinity-tag-reactive moiety and affinity-tagged
antisense rRNA molecules are generated.
[0011] Work conducted during the development of embodiments of the
present invention found that the relative number of the affinity
tags per given number of nucleobases in the affinity-tagged
antisense rRNA molecules is important for generating a composition
comprising affinity-tagged antisense rRNA molecules that can be
used to remove >95% of the at least one rRNA molecules from a
sample, and that the number of the affinity tags per given number
of nucleobases in the affinity-tagged antisense rRNA molecules
generated using the method varies based on the amount of the
affinity-tag-reactive moieties present in the antisense rRNA
molecules generated in step b), the properties and concentration of
the affinity tag reagent, the reaction conditions, and the reaction
time. Thus, in some embodiments of this method, 100% of the
ribonucleoside-5'-triphosphate molecules comprising one particular
nucleobase have an affinity-tag-reactive moiety. In some other
embodiments of this method, the ribonucleoside-5'-triphosphates
includes at least one pair of ribonucleoside-5'-triphosphates
complementary to the same nucleobase, one of which pair comprises
an affinity-tag-reactive moiety (e.g., UTP having an allylamino
reactive group on the 5 position of uridine or "AA-UTP") and the
other of which pair does not comprise an affinity tag. In some
embodiments of this method, at least about 50% of the
ribonucleoside-5'-triphosphate molecules comprising the at least
one pair of ribonucleoside-5'-triphosphates have an
affinity-tag-reactive moiety and about 50% of the
ribonucleoside-5'-triphosphate molecules comprising the pair do not
have an affinity-tag-reactive moiety. In some embodiments of this
method, at least about 75% of the ribonucleoside-5'-triphosphate
molecules comprising the at least one pair of
ribonucleoside-5'-triphosphates have an affinity-tag-reactive
moiety and 25% of the ribonucleoside-5'-triphosphate molecules
comprising the pair do not have an affinity-tag-reactive moiety. In
some embodiments of this method, the affinity tag reagent is a
biotinylation reagent (e.g., Biotin-X-X-NHS) and the affinity tag
comprises biotin. In some embodiments, the biotin is joined via a
spacer arm to the nucleobase (e.g., a spacer arm joined to the
allylamino group on the 5 position of uridine, e.g., from
incorporation of AA-UTP).
[0012] In further embodiments, the present invention provides a
method for generating a composition comprising antisense rRNA
molecules comprising: a) generating double-stranded DNA molecules
comprising an RNA polymerase promoter that directs RNA synthesis of
antisense RNA corresponding to all of at least one rRNA molecule
selected from among 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules;
and b) contacting the double-stranded DNA molecules comprising the
RNA polymerase promoter with an RNA polymerase and
ribonucleoside-5'-triphosphates complementary to all of the
nucleobases, including at least one pair of
ribonucleoside-5'-triphosphates complementary to the same
nucleobase, one of which pair comprises an affinity tag and the
other of which pair does not comprise an affinity tag, and
incubating under conditions wherein affinity-tagged antisense rRNA
molecules are generated corresponding to substantially all of the
sequence of the at least one rRNA molecule, wherein the affinity
tags are present at a ratio of at least two affinity tags per
hundred nucleobases of the affinity-tagged antisense rRNA molecules
(e.g., based on fluorescence quantification of 200 ng of
affinity-tagged antisense rRNA according to the instructions
supplied with the Fluorescence Biotin Quantitation Kit, Pierce
Biotechnology, Rockford, Ill.; Cat. #: Thermo 46610) after
digestion of the affinity-tagged antisense rRNA with RNase 1 at
36.degree. C. for 45 min and heat inactivation of the enzyme
according to the RNase 1 literature provided by the manufacturer,
EPICENTRE Biotechnologies, Madison, Wis.). In some other
embodiments of this method, the affinity tags are present at a
ratio of at least about three to five affinity tags per hundred
nucleobases of the affinity-tagged antisense rRNA molecules (e.g.,
based on the fluorescence data obtained using the Pierce
Fluorescence Biotin Quantitation Kit). In some other embodiments of
this method, the affinity tags are present at a ratio of at least
about four to six affinity tags per hundred nucleobases of the
affinity-tagged antisense rRNA molecules (e.g., based on the
fluorescence data obtained using the Pierce Fluorescence Biotin
Quantitation Kit). In some other embodiments of this method, the
affinity tags are present at a ratio of at least about six to eight
affinity tags per hundred nucleobases of the affinity-tagged
antisense rRNA molecules (e.g., based on the fluorescence data
obtained using the Pierce Fluorescence Biotin Quantitation Kit). In
some embodiments of this method wherein the at least one rRNA
molecule is selected from among 28S, 26S, 25S, and 18S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S and 16S prokaryotic rRNA molecules, at
least about 35% to about 50% of the ribonucleoside-5'-triphosphates
comprising the at least one pair of ribonucleoside-5'-triphosphates
complementary to the same nucleobase comprise the affinity tag
(e.g., an affinity tag comprising biotin; e.g., biotin-16-UTP) and
the affinity tags are present in the affinity-tagged antisense rRNA
molecules generated at a ratio of at least about two to eight
affinity tags per hundred nucleobases (e.g., based on the
fluorescence data obtained using the Pierce Fluorescence Biotin
Quantitation Kit). In some embodiments of this method wherein the
at least one rRNA molecule is selected from among 5.8S and 5S
eukaryotic rRNA molecules and 5S prokaryotic rRNA molecules, at
least about 60% to about 75% of the ribonucleoside-5'-triphosphates
comprising the at least one pair of ribonucleoside-5'-triphosphates
complementary to the same nucleobase comprise the affinity tag
(e.g., an affinity tag comprising biotin; e.g., biotin-16-UTP) and
the affinity tags are present in the affinity-tagged antisense rRNA
molecules generated at a ratio of at least about two to eight
affinity tags per hundred nucleobases (e.g., based on the
fluorescence data obtained using the Pierce Fluorescence Biotin
Quantitation Kit).
[0013] In further embodiments, the present invention provides a
method for generating a composition comprising antisense rRNA
molecules comprising: a) generating double-stranded DNA molecules
comprising an RNA polymerase promoter that directs RNA synthesis of
antisense RNA corresponding to all of at least one rRNA molecule
selected from among 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules; b)
contacting the double-stranded DNA molecules comprising the RNA
polymerase promoter with an RNA polymerase and
ribonucleoside-5'-triphosphates complementary to all of the
nucleobases, including at least one ribonucleoside-5'-triphosphate
that comprises an affinity-tag-reactive moiety (e.g., an allylamino
group on the 5 position of uridine), and incubating under
conditions wherein antisense rRNA molecules comprising the
affinity-tag-reactive moiety are generated, which antisense rRNA
molecules correspond to substantially all of the sequence of the at
least one rRNA molecules; and c) contacting the antisense rRNA
molecules comprising the affinity-tag-reactive moiety with a
quantity of an affinity tag reagent (e.g., Biotin-X-X-NHS,
EPICENTRE Biotechnologies, Madison, Wis.) under conditions wherein
the affinity tag reagent reacts with the affinity-tag-reactive
moieties in the antisense rRNA molecules comprising the
affinity-tag-reactive moiety and affinity-tagged antisense rRNA
molecules are generated, wherein the affinity tags are present at a
ratio of at least about two affinity tags per hundred nucleobases
of the affinity-tagged antisense rRNA molecules (e.g., based on
fluorescence quantification of RNase 1-digested affinity-tagged
antisense rRNA molecules (200 nanograms) using the Fluorescence
Biotin Quantitation Kit from Pierce Biotechnology, Rockford, Ill.).
In some other embodiments of this method, the affinity tags are
present at a ratio of at least about three to five affinity tags
per hundred nucleobases of the affinity-tagged antisense rRNA
molecules (e.g., based on the fluorescence data obtained using the
Pierce Fluorescence Biotin Quantitation Kit). In some other
embodiments of this method, the affinity tags are present at a
ratio of at least about four to six affinity tags per hundred
nucleobases of the affinity-tagged antisense rRNA molecules (e.g.,
based on the fluorescence data obtained using the Pierce
Fluorescence Biotin Quantitation Kit). In some other embodiments of
this method, the affinity tags are present at a ratio of at least
about six to eight affinity tags per hundred nucleobases of the
affinity-tagged antisense rRNA molecules (e.g., based on the
fluorescence data obtained using the Pierce Fluorescence Biotin
Quantitation Kit).
[0014] In some embodiments of any of the methods herein for
generating affinity-tagged antisense rRNA molecules, the affinity
tag comprises biotin. In some embodiments, the biotin is joined via
a spacer arm to the 5 position of uridine. In some embodiments of
any of these methods, the RNA polymerase is selected from among T7
RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. In some
embodiments of any of these methods, wherein the at least one rRNA
molecule is selected from among eukaryotic cytoplasmic 25S, 26S,
and 28S rRNA molecules, the RNA polymerase is SP6 RNA polymerase.
In some embodiments of any of these methods, wherein the at least
one rRNA molecule is a prokaryotic 23S rRNA, the RNA polymerase is
SP6 RNA polymerase.
[0015] In certain embodiments of any of the above methods for
generating affinity-tagged antisense rRNA molecules, the methods
further comprise, after generating the antisense rRNA molecules,
contacting the double-stranded DNA molecules comprising the RNA
polymerase promoter with a DNase enzyme under conditions wherein
the double-stranded DNA molecules comprising the RNA polymerase
promoter are digested.
[0016] In some embodiments of any of the methods for generating
affinity-tagged antisense rRNA molecules, step a) of the method
(comprising "generating double-stranded DNA molecules comprising an
RNA polymerase promoter that directs RNA synthesis of antisense RNA
corresponding to all of at least one rRNA molecule selected from
among 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA
molecules, 12S and 16S eukaryotic mitochondrial rRNA molecules, and
23S, 16S, and 5S prokaryotic rRNA molecules") comprises either: i)
incubating the at least one rRNA molecule with a DNA polymerase and
at least one primer pair under conditions wherein double-stranded
DNA molecules that comprise an RNA polymerase promoter that is
capable of directing RNA synthesis of antisense RNA corresponding
to all of the at least one rRNA molecule are generated, wherein
each said at least one primer pair comprises a forward primer and a
reverse primer, wherein the reverse primer anneals to the at least
one rRNA molecule and has a 5' portion that exhibits the sequence
of one strand of an RNA polymerase promoter and the forward primer
anneals to the DNA generated from the reverse primer; or: ii)
incubating the at least one rRNA molecule with a DNA polymerase and
at least one primer pair under conditions wherein double-stranded
DNA molecules are generated, wherein each said at least one primer
pair comprises a forward primer and a reverse primer, wherein the
reverse primer primes DNA synthesis after annealing to the at least
one rRNA molecule and the forward primer primes DNA synthesis after
annealing to the DNA generated from DNA polymerase extension of the
reverse primer, and then ligating said double-stranded DNA
molecules generated from each at least one primer pair into a DNA
vector that comprises an RNA polymerase promoter, wherein RNA
polymerase promoter is capable of directing synthesis of antisense
RNA that is complementary to the at least one rRNA molecule using
said double-stranded DNA that is ligated into said DNA vector as a
template.
[0017] In some embodiments of any of the methods for generating
affinity-tagged antisense rRNA molecules, step a) of the method
(comprising "generating double-stranded DNA molecules comprising an
RNA polymerase promoter that directs RNA synthesis of antisense RNA
corresponding to all of at least one rRNA molecule selected from
among 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA
molecules, 12S and 16S eukaryotic mitochondrial rRNA molecules, and
23S, 16S, and 5S prokaryotic rRNA molecules") comprises obtaining a
genomic DNA fragment that encodes the at least one rRNA molecule
and either: i) contacting said genomic DNA fragment with a DNA
polymerase and at least one primer pair, wherein the first primer
of each of said at least one primer pair has a 5' portion that
exhibits the sequence of one strand of an RNA polymerase promoter
and a 3' portion that is complementary to a portion of a first
strand of the genomic DNA fragment and the second primer of each of
said at least one primer pair is complementary to a portion of the
second strand of the genomic DNA fragment, and incubating under
conditions wherein RNA polymerase promoter-containing
double-stranded DNA copies of at least a portion of the genomic DNA
fragment are generated, wherein the RNA polymerase promoter is
capable of directing RNA synthesis of antisense RNA corresponding
to all of the at least one rRNA molecule; or: ii) ligating said
genomic DNA fragment or a PCR amplification product thereof into a
DNA vector that comprises an RNA polymerase promoter to obtain a
genomic clone, wherein the RNA polymerase promoter in said genomic
clone is capable of directing synthesis of antisense RNA that is
complementary to the at least one rRNA molecule using said
double-stranded DNA that is ligated into said DNA vector as a
template.
[0018] In some embodiments of the methods for generating
affinity-tagged antisense rRNA molecules and of the compositions
generated using the method, the affinity-tagged antisense rRNA
molecules do not exhibit any rRNA internal transcribed spacer
sequences. In some other embodiments, the affinity-tagged antisense
rRNA molecules exhibit internal transcribed spacer sequences
selected from among: i) a sequence exhibited by the ITS1 rRNA
spacer region, which is located between a eukaryotic 18S rRNA gene
and a eukaryotic 5.8S rRNA gene; ii) a sequence exhibited by the
ITS2 rRNA spacer region, which is located between a eukaryotic 5.8S
rRNA gene and a eukaryotic 28S rRNA gene; iii) a sequence exhibited
by a prokaryotic 16S-23S ITS; and iv) a sequence exhibited by a
prokaryotic 23S-5S rRNA ITS.
[0019] In some embodiments, the present invention provides a
composition comprising affinity-tagged antisense rRNA molecules
that are bound to a binding matrix comprising affinity-tag-binding
molecules, wherein the affinity-tagged antisense rRNA molecules,
alone or in combination, exhibit sequences corresponding to
substantially all of at least one full-length rRNA molecule
selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA
molecules.
[0020] A composition comprising affinity-tagged antisense rRNA
molecules that are bound to a binding matrix comprising
affinity-tag-binding molecules is generated by incubating the
composition comprising affinity-tagged antisense rRNA molecules
with a binding matrix comprising affinity-tag-binding molecules
under binding conditions, wherein the affinity tag binds to the
affinity-tag-binding molecules that are attached to the matrix or
solid support (e.g., microparticles) to form a specific binding
pair. In some embodiments, this method further comprises the step
of washing the composition under conditions wherein affinity-tagged
antisense rRNA molecules that are not specifically bound to the
binding matrix are removed, thereby generating a purified
composition comprising affinity-tagged antisense rRNA molecules
that are bound to the binding matrix.
[0021] In particular embodiments of this aspect of the invention,
the composition comprising affinity-tagged antisense rRNA molecules
that are bound to a binding matrix can be any composition of
affinity-tagged antisense rRNA molecules described herein and can
be generated using any method for generating a composition
comprising antisense rRNA molecules described herein.
[0022] In one particular embodiment of this composition, the
affinity-tagged antisense rRNA molecules comprise biotin as the
affinity tag, wherein the biotin is joined to at least about two
nucleobases per hundred nucleobases of the antisense rRNA
molecules, and the binding matrix comprises a microparticle to
which an affinity-tag-binding molecule comprising streptavidin or
avidin is attached. In some other embodiments of this composition,
the affinity-tagged antisense rRNA molecules comprise biotin as the
affinity tag, wherein the biotin is joined to at least about two to
four nucleobases per hundred nucleobases of the antisense rRNA
molecules, or to at least three to five nucleobases per hundred
nucleobases of the antisense rRNA molecules, or to at least four to
six nucleobases per hundred nucleobases of the antisense rRNA
molecules, or to at least six to eight nucleobases per hundred
nucleobases of the antisense rRNA molecules.
[0023] In some embodiments of the invention, any of the
compositions comprising affinity-tagged antisense rRNA molecules,
including any of the compositions comprising affinity-tagged
antisense rRNA molecules that are bound to a binding matrix, is
used in a method of the invention for generating a rRNA-depleted
sample or for isolating substantially all of the RNA molecules
that, either alone or in combination, exhibit the sequence of at
least one full-length rRNA molecule.
[0024] In some embodiments, the present invention provides methods
for generating a rRNA-depleted sample from an initial sample
comprising: a) providing i) an initial sample comprising RNA
molecules, wherein the RNA molecules comprise rRNA molecules and at
least one non-rRNA RNA molecule of interest; ii) a composition
comprising affinity-tagged antisense rRNA molecules that, alone or
in combination, exhibit sequences corresponding to substantially
all of at least one full-length rRNA molecule selected from: 25S,
26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules,
12S and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S,
and 5S prokaryotic rRNA molecules; and iii) a binding matrix (e.g.,
microparticles) comprising affinity-tag-binding molecules; b)
contacting the initial sample with the composition under conditions
such that at least some of the affinity-tagged antisense rRNA
molecules and at least some of the rRNA molecules form
double-stranded rRNA hybrids thereby generating a treated sample;
c) contacting the treated sample with the binding matrix under
conditions such that at least a portion of the double-stranded rRNA
hybrids bind to the binding matrix and are removed from the treated
sample, thereby generating a rRNA-depleted sample, wherein the
rRNA-depleted sample is substantially free of rRNA sequences
exhibited by the at least one rRNA molecule and comprises
substantially all of the at least one non-rRNA RNA molecule of
interest present in the initial sample (e.g., at least >90% . .
. , >95% . . . , >98% . . . , >99% . . . , >99.8% . . .
, or >99.9% of the at least one non-rRNA RNA molecule of
interest present in the initial sample).
[0025] In certain embodiments, one or more steps are repeated
and/or additional methods are performed to generate the
rRNA-depleted sample. In particular embodiments, the methods are
performed with repeated rounds of subtraction in order to generate
the rRNA-depleted sample. In particular embodiments, at least a
portion of the RNA molecules in the initial sample are highly
fragmented, and wherein the rRNA-depleted sample is at least 50% .
. . 60% . . . 70% . . . 80% . . . 90.0% . . . 95% . . . 98% . . .
99% . . . or 100% free of rRNA sequences exhibited by the at least
one rRNA molecule.
[0026] In certain embodiments, the present invention provides
methods for generating a rRNA-depleted sample from an initial
sample comprising: a) providing: i) an initial sample comprising
RNA molecules, wherein the RNA molecules comprise rRNA molecules
and at least one non-rRNA RNA molecule of interest; ii) a
composition comprising affinity-tagged antisense rRNA molecules
that, alone or in combination, are complementary to substantially
all of the sequence exhibited by the at least one rRNA molecule
selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules;
and iii) a binding matrix (e.g., microparticles) comprising
affinity-tag-binding molecules; b) contacting the initial sample
with the composition under conditions such that at least some of
the affinity-tagged antisense rRNA molecules and at least some of
the rRNA molecules form double-stranded rRNA hybrids thereby
generating a treated sample; c) contacting the treated sample with
the binding matrix under conditions such that at least a portion of
the double-stranded rRNA hybrids bind to the binding matrix and are
removed from the treated sample, thereby generating an
rRNA-depleted sample, wherein the rRNA-depleted sample comprises
substantially all (e.g., >90% . . . , >95% . . . , >98% .
. . , >99% . . . , >99.8% . . . , or >99.9%) of the at
least one non-rRNA RNA molecule of interest present in the initial
sample and is substantially free (e.g., >95% . . . , >98% . .
. , >99% . . . , >99.8% . . . , or >99.9% free) of rRNA
sequences exhibited by the at least one rRNA molecule present in
the initial sample.
[0027] In additional embodiments, the present invention provides
methods for generating a rRNA-depleted sample from an initial
sample comprising: a) providing; i) an initial sample comprising
RNA molecules, wherein the RNA molecules comprise rRNA molecules
and at least one non-rRNA RNA molecules of interest; ii) a
composition comprising affinity-tagged antisense rRNA molecules
complementary to substantially all of the sequence exhibited by the
at least one rRNA molecule selected from: 25S, 26S, 28S, 18S, 5.8S,
and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules, wherein the affinity tags are present
on the antisense rRNA molecules at a ratio of at least about two to
at least four affinity-tags per hundred nucleobases of the
antisense rRNA molecules; and iii) a binding matrix comprising
affinity-tag-binding molecules; b) contacting the initial sample
with the composition under conditions such that at least some of
the affinity-tagged antisense rRNA molecules and at least some of
the rRNA molecules form double-stranded rRNA hybrids thereby
generating a treated sample; c) contacting the treated sample with
the binding matrix under conditions wherein at least a portion of
the double-stranded rRNA hybrids bind to the binding matrix and are
removed from the treated sample, thereby generating a rRNA-depleted
sample, wherein the rRNA-depleted sample comprises substantially
all (e.g., >90% . . . , >95% . . . , >98% . . . , >99%
. . . , >99.8% . . . , or >99.9%) of the at least one
non-rRNA RNA molecule of interest present in the initial sample and
is substantially free (e.g., >95% . . . , >98% . . . ,
>99% . . . , >99.8% . . . , or >99.9% free) of molecules
that, either alone or in combination, exhibit the sequences within
the at least one rRNA molecule present in the initial sample.
[0028] In certain embodiments, the present invention provides
methods for generating a rRNA-depleted sample from an initial
sample comprising: a) providing; i) an initial sample comprising
RNA molecules, wherein the RNA molecules comprise rRNA molecules
and at least one non-rRNA RNA molecule of interest; ii) a
composition comprising affinity-tagged antisense rRNA molecules
complementary to substantially all of the sequence exhibited by the
at least one rRNA molecule selected from: 25S, 26S, 28S, 18S, 5.8S,
and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules; and iii) a binding matrix (e.g.,
microparticles) comprising affinity-tag-binding molecules; b)
contacting the initial sample with the composition under conditions
such that at least some of the affinity-tagged antisense rRNA
molecules and at least some of the rRNA molecules form
double-stranded rRNA hybrids thereby generating a treated sample;
c) contacting the treated sample with the binding matrix under
conditions such that at least a portion of the double-stranded rRNA
hybrids bind to the binding matrix and are removed from the treated
sample, thereby generating a rRNA-depleted sample, wherein the
rRNA-depleted sample comprises substantially all (e.g., >90% . .
. , >95% . . . , >98% . . . , >99% . . . , >99.8% . . .
, or >99.9%) of the at least one non-rRNA RNA molecule of
interest present in the initial sample) and wherein, the
rRNA-depleted sample is either: i) .gtoreq.99.0% free (e.g.,
>99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,
99.9%, 99.95%, or 99.99% free) of RNA molecules that, either alone
or in combination, exhibit a sequence within the at least one rRNA
molecule present in the initial sample, or ii) contains
undetectable levels of the at least one rRNA molecules from the
initial sample as measured using agarose gel electrophoresis and
ethidium bromide staining.
[0029] In some embodiments, the present invention provides methods
for generating a rRNA-depleted sample from an initial sample
comprising: a) providing i) an initial sample comprising RNA
molecules, wherein the RNA molecules comprise rRNA molecules and at
least one non-rRNA RNA molecule of interest; and ii) a composition
comprising affinity-tagged antisense rRNA molecules that are bound
to a binding matrix (e.g., microparticles comprising
affinity-tag-binding molecules), wherein the affinity-tagged
antisense rRNA molecules, alone or in combination, exhibit
sequences corresponding to substantially all of at least one
full-length rRNA molecule selected from: 25S, 26S, 28S, 18S, 5.8S,
and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules; b) contacting the initial sample with
the composition under conditions such that at least some of the
affinity-tagged antisense rRNA molecules in the composition and at
least some of the rRNA molecules form double-stranded rRNA hybrids
that are bound to the binding matrix, thereby generating a treated
sample; and c) removing the binding matrix to which the
double-strand rRNA hybrids are bound, thereby generating a
rRNA-depleted sample, wherein the rRNA-depleted sample is: i)
substantially free of rRNA sequences exhibited by the at least one
rRNA molecule and comprises substantially all of the at least one
non-rRNA RNA molecule of interest present in the initial sample
(e.g., at least >90% . . . , >95% . . . , >98% . . . ,
>99% . . . , >99.8% . . . , or >99.9% of the at least one
non-rRNA RNA molecule of interest present in the initial sample),
and ii) either, substantially free (e.g., >95% . . . , >98% .
. . , >99% . . . , >99.8% . . . , or >99.9% free) of rRNA
sequences exhibited by the at least one rRNA molecule present in
the initial sample, or, contains undetectable levels of the at
least one rRNA molecules from the initial sample as measured using
agarose gel electrophoresis and ethidium bromide staining. In
particular embodiments of this method of the invention, the
composition comprising affinity-tagged antisense rRNA molecules
that are bound to a binding matrix is any composition of
affinity-tagged antisense rRNA molecules described herein and/or is
generated using any method for generating a composition comprising
antisense rRNA molecules described herein.
[0030] In particular embodiments of any of the methods for
generating an rRNA-depleted sample, the rRNA-depleted sample is
98.0% free of rRNA molecules that exhibit sequences within the at
least one rRNA molecule (e.g., 98.0% free, 98.5% free, 99.0% free,
99.5% free, 99.7% free, 99.9% free, 99.99% free, or 100% free). In
other embodiments, the at least one rRNA molecule includes both the
28S rRNA and the 18S rRNA, and wherein the rRNA-depleted sample is
99.0% free (e.g., 99.5% free) of rRNA molecules that exhibit
sequences within the 28S rRNA gene and the 18S rRNA gene. In other
embodiments, the rRNA-depleted sample contains undetectable levels
of rRNA molecules that exhibit sequences within the at least one
rRNA molecule (e.g., undetectable as measured using agarose gel
electrophoresis and ethidium bromide staining). In some embodiments
of the method, the composition comprising affinity-tagged antisense
rRNA molecules does not exhibit any rRNA internal transcribed
spacer sequences. In some other embodiments of the method, the
composition comprising the antisense rRNA molecules exhibits
internal transcribed spacer sequences selected from among: i) a
sequence exhibited by the ITS1 rRNA spacer region, which is located
between a eukaryotic 18S rRNA gene and a eukaryotic 5.8S rRNA gene;
ii) a sequence exhibited by the ITS2 rRNA spacer region, which is
located between a eukaryotic 5.8S rRNA gene and a eukaryotic 28S
rRNA gene; iii) a sequence exhibited by a prokaryotic 16S-23S ITS;
and iv) a sequence exhibited by a prokaryotic 23S-5S rRNA ITS. In
particular embodiments of the methods for generating rRNA-depleted
samples or for isolating rRNA from a sample, step b) is performed
using a composition comprising antisense rRNA molecules that do not
exhibit any rRNA internal transcribed spacer sequences, whereas, in
other embodiments, step b) is performed using a composition
comprising affinity-tagged antisense rRNA molecules that exhibit
one or more of the internal transcribed spacer sequences.
[0031] In some embodiments of any of the methods for generating a
rRNA-depleted sample from an initial sample wherein the initial
sample comprises RNA molecules that comprise rRNA molecules and at
least one non-rRNA RNA molecule of interest, the RNA molecules in
the initial sample are fragmented (e.g., from an FFPE or other
sample comprising degraded RNA molecules, or from a sample wherein
the RNA molecules are deliberated degraded (e.g. by incubation in
the presence of heat and Mg2+) prior to being provided in the
initial sample). In some embodiments of any of the methods of the
invention for generating a rRNA-depleted sample, the at least one
non-rRNA RNA molecule of interest comprises a multiplicity of
non-rRNA molecules of interest. In some embodiments, the at least
one non-rRNA RNA molecule of interest comprises substantially all
of the non-rRNA RNA molecules present in the initial sample (e.g.,
including both the intact and fragmented non-rRNA RNA molecules
present in the initial sample). In some embodiments, the at least
one non-rRNA RNA molecule of interest comprises substantially all
of the eukaryotic mRNA molecules present in the initial sample. In
some embodiments, the at least one non-rRNA RNA molecule of
interest comprises substantially all of the non-rRNA RNA molecules
comprising the transcriptome (minus the at least one rRNA molecule)
from one or multiple eukaryotic cells, tissues, organs, or
organisms (e.g., for use in making sequencing templates or labeled
target nucleic acid, e.g., for analysis of the expression or
relative expression of said at least one non-rRNA RNA molecule of
interest by digital expression analysis or microarray analysis,
respectively). In some embodiments, the at least one non-rRNA RNA
molecule of interest comprises the non-rRNA RNA molecules
comprising a transcriptome (minus the at least one rRNA molecule)
of one or multiple eukaryotic cells, tissues, organs, or organisms
(e.g., for use in making sequencing templates or labeled target
nucleic acid, e.g., for analysis of the expression or relative
expression of all of said non-rRNA RNA molecules by digital
expression analysis or microarray analysis, respectively, e.g., for
medical or agricultural analysis). In some embodiments, the at
least one non-rRNA RNA molecule of interest comprises a subfraction
of the non-rRNA RNA molecules comprising the transcriptome (minus
the at least one rRNA molecules) of one or multiple cells, tissues,
organs, or organisms (e.g., a subfraction selected from among mRNA
molecules, miRNA molecules, ncRNA molecules, piwiRNA, snRNA, etc.)
(e.g., for use in making sequencing templates or labeled target
nucleic acid, e.g., for analysis of the expression or relative
expression of said subfraction of non-rRNA RNA molecules, e.g., by
digital expression analysis or microarray analysis, respectively,
e.g., for medical or agricultural analysis). In some embodiments,
the at least one non-rRNA RNA molecule of interest comprises
substantially all of the non-rRNA RNA molecules comprising a
transcriptome (minus the at least one rRNA molecule) of one or
multiple prokaryotic cells or of one or multiple cells comprising
both eukaryotic and prokaryotic cells. In some embodiments, the at
least one non-rRNA RNA molecule of interest comprises or consists
of one or multiple RNA molecules that exhibit specific nucleic acid
sequences (e.g., wherein the presence or absence or the quantity of
said one or multiple RNA molecules that exhibit the specific
nucleic acid sequences is used to detect a pathogen or a medical
condition, e.g., for screening (e.g., for screening for the
presence of a a pathogen in water, on a surface, such as a hospital
surface, etc.), or e.g., for diagnostic or theranostic assay (e.g.,
for diagnosing or monitoring the quantity of a pathogen, or the
status of a disease or medical condition for deciding on a therapy
or treatment).
[0032] In some embodiments of any of the methods of the invention
for generating a rRNA-depleted sample, the method further comprises
using the rRNA-depleted sample or the at least one non-rRNA RNA
molecule of interest contained therein for further analysis or use.
In some embodiments, the method further comprises using the at
least one non-rRNA RNA molecule of interest as part of a method for
generating templates for next-generation DNA sequencing (e.g., for
digital expression analysis or RNA-Seq, miRNA profiling, etc.). In
some embodiments of the method for generating a rRNA-depleted
sample, the method further comprises using the at least one
non-rRNA RNA molecule of interest as part of a method for
generating labeled target nucleic acid molecules for hybridization
to probes of an array or microarray on a porous or non-porous
surface (e.g., to probes on an array or microarray, a dot blot,
etc.). In some embodiments of the method for generating a
rRNA-depleted sample, the method further comprises using the
rRNA-depleted sample for performing a diagnostic or theranostic
assay to detect for the presence of the at least one non-rRNA RNA
molecule of interest (e.g., wherein the presence or quantity of
said at least one non-rRNA RNA molecule of interest is indicative
of the presence or status of a health or disease state). In some
embodiments of the method for generating a rRNA-depleted sample,
the method further comprises amplifying the at least one non-rRNA
RNA molecule of interest for further analysis or use. In some
embodiments of the method for generating a rRNA-depleted sample,
the method further comprises using the at least one non-rRNA RNA
molecule of interest or an amplification product thereof for
transfection of a eukaryotic cell (e.g., an antigen-presenting cell
(APC), such as a dendritic cell, a macrophage, or an artificial APC
for immunotherapeutic use). In some embodiments, the at least one
non-rRNA RNA molecule of interest or an amplification product
thereof is used for transfection of a human or animal cell from the
same individual from whom the at least one non-rRNA RNA molecule of
interest was obtained (e.g., to make a vaccine comprising the
APC-transfected cell for immunotherapeutic use to treat a disease,
e.g., cancer, in a human or animal individual). In some
embodiments, the at least one non-rRNA RNA molecule of interest or
an amplification product thereof is used for transfection of a cell
from a different human or animal than the cell from whom the at
least one non-rRNA RNA molecule of interest was obtained (e.g., to
make a vaccine comprising the APC-transfected cell for
immunotherapeutic use to treat a disease, e.g., cancer, in a human
or animal individual). In certain embodiments, the RNA vaccine is
made using the at least one non-rRNA RNA of interest from a first
individual and the RNA vaccine is used to vaccinate a second
individual. In some embodiments of the method for generating a
rRNA-depleted sample, the method further comprises using the at
least one non-rRNA RNA molecule of interest or an amplification
product thereof to manufacture an RNA vaccine for therapeutic use.
In some embodiments of the method, the at least one non-rRNA RNA
molecule of interest or a sense RNA amplification product thereof
is used to manufacture an RNA vaccine that is used to directly
inoculate a patient for therapeutic use. In some embodiments, the
RNA vaccine is made using the at least one non-rRNA RNA of interest
from a cell, tissue, or organ from first individual and is the RNA
vaccine is administered to said first individual as an
immunotherapeutic treatment. In some embodiments, the RNA vaccine
is made using the at least one non-rRNA RNA of interest from a
first individual and the RNA vaccine is used to vaccinate a second
individual.
[0033] In still other embodiments, the invention provides methods
for using the composition comprising affinity-tagged antisense rRNA
molecules for isolating substantially all of the RNA molecules
that, either alone or in combination, exhibit a sequence within at
least one full-length rRNA molecule selected from among 25S, 26S,
28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules, 12S
and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and
5S prokaryotic rRNA molecules.
[0034] Thus, in some embodiments, the present invention provides
methods for isolating substantially all of the RNA molecules that,
either alone or in combination, exhibit the sequence within at
least one full-length rRNA molecule, the method comprising: a)
providing i) an initial sample comprising RNA molecules, wherein
the RNA molecules comprise rRNA molecules and non-rRNA RNA
molecules; ii) a composition comprising affinity-tagged antisense
rRNA molecules that exhibits sequences corresponding to
substantially all of at least one full-length rRNA molecule
selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules;
and iii) a binding matrix (e.g., microparticles) comprising
affinity-tag-binding molecules; b) contacting the initial sample
with the composition under conditions such that at least some of
the affinity-tagged antisense rRNA molecules and at least some of
the rRNA molecules form double-stranded rRNA hybrids thereby
generating a treated sample; c) contacting the treated sample with
the binding matrix under conditions such that at least a portion of
the double-stranded rRNA hybrids bind to the binding matrix; d)
removing the binding matrix to which are bound the double-stranded
rRNA hybrids comprising the affinity-tagged antisense rRNA
molecules and the at least some of the rRNA molecules from the
treated sample; and e) incubating the binding matrix in a solution
under conditions wherein the at least some rRNA molecules from the
treated sample are released into the solution, thereby isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit a sequence within at least one full-length
rRNA molecule present in the initial sample (e.g., at least >95%
. . . , >98% . . . , >99% . . . , >99.8% . . . , or
>99.9% of the at least one full-length rRNA molecules present in
the initial sample).
[0035] In some embodiments, the present invention provides methods
for isolating substantially all of the RNA molecules that, either
alone or in combination, exhibit the sequence within at least one
full-length rRNA molecule, the method comprising: a) providing i)
an initial sample comprising RNA molecules, wherein the RNA
molecules comprise rRNA molecules and non-rRNA RNA molecules; ii) a
composition comprising affinity-tagged antisense rRNA molecules
that exhibits sequences corresponding to substantially all of at
least one full-length rRNA molecule selected from: 25S, 26S, 28S,
18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules, 12S and
16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules; and iii) a binding matrix (e.g.,
microparticles) comprising affinity-tag-binding molecules; b)
contacting the initial sample with the composition under conditions
such that at least some of the affinity-tagged antisense rRNA
molecules and at least some of the rRNA molecules form
double-stranded rRNA hybrids thereby generating a treated sample;
c) contacting the treated sample with the binding matrix under
conditions such that at least a portion of the double-stranded rRNA
hybrids bind to the binding matrix; d) removing the binding matrix
to which are bound the double-stranded rRNA hybrids comprising the
affinity-tagged antisense rRNA molecules and the at least some of
the rRNA molecules from the treated sample; and e) incubating the
binding matrix in a solution under conditions wherein the at least
some rRNA molecules from the treated sample are released into the
solution, thereby generating an isolated rRNA sample comprising
substantially all of the RNA molecules that, either alone or in
combination, exhibit a sequence within at least one full-length
rRNA molecule present in the initial sample (e.g., at least >90%
. . . , >95% . . . , >98% . . . , >99% . . . , >99.8% .
. . , or >99.9% of the at least one full-length rRNA molecule
present in the initial sample) and wherein the isolated rRNA sample
is substantially free (e.g., at least >90% . . . , >95% . . .
, >98% . . . , >99% . . . , >99.8% . . . , or >99.9%
free) of the non-rRNA RNA molecules present in the initial
sample.
[0036] In some embodiments, the present invention provides methods
for isolating substantially all of the RNA molecules that, either
alone or in combination, exhibit the sequence within at least one
full-length rRNA molecule, the method comprising: a) providing i)
an initial sample comprising RNA molecules, wherein the RNA
molecules comprise rRNA molecules and non-rRNA RNA molecules; ii) a
composition comprising affinity-tagged antisense rRNA molecules
that exhibits sequences corresponding to substantially all of at
least one full-length rRNA molecule selected from: 25S, 26S, 28S,
18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules, 12S and
16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules; and iii) a binding matrix (e.g.,
microparticles) comprising affinity-tag-binding molecules; b)
contacting the initial sample with the composition under conditions
such that at least some of the affinity-tagged antisense rRNA
molecules and at least some of the rRNA molecules form
double-stranded rRNA hybrids thereby generating a treated sample;
c) contacting the treated sample with the binding matrix under
conditions such that at least a portion of the double-stranded rRNA
hybrids bind to the binding matrix; d) removing the binding matrix
to which are bound the double-stranded rRNA hybrids comprising the
affinity-tagged antisense rRNA molecules and the at least some of
the rRNA molecules from the treated sample; and e) incubating the
binding matrix in a solution under conditions wherein the at least
some rRNA molecules from the treated sample are released into the
solution, thereby generating an isolated rRNA sample comprising
substantially all of the RNA molecules that, either alone or in
combination, exhibit a sequence within at least one full-length
rRNA molecule present in the initial sample (e.g., at least >90%
. . . , >95% . . . , >98% . . . , >99% . . . , >99.8% .
. . , or >99.9% of the at least one full-length rRNA molecule
present in the initial sample) and wherein, either i) the isolated
rRNA sample is substantially free (e.g., at least >90% . . . ,
>95% . . . , >98% . . . , >99% . . . , >99.8% . . . ,
or >99.9% free) of the non-rRNA RNA molecules present in the
initial sample, or ii) contains undetectable levels of non-rRNA RNA
from the initial sample as measured using agarose gel
electrophoresis and ethidium bromide staining.
[0037] In some embodiments of the methods for isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit the sequence within at least one full-length
rRNA molecule, the isolated rRNA sample is from an initial sample
(e.g., comprising a biological specimen, including a medical
specimen, such as saliva, sputum, feces, urine, or a cell, tissue,
or organ sample, an environmental sample, a metagenomic sample, or
any other type of sample which may contain the RNA of interest),
whether the sample is unprepared or prepared (e.g., by fixation
using a solution, e.g., formalin or ethanol; mounting on a slide;
or using other procedures known in the art), and the isolated rRNA
is analyzed to determine the genera, species or strains of origin,
thereby indicating what particular genera, species or strains were
present in the initial sample, and therefore, in the particular
specimen or environment from which the sample was collected. For
example, in some embodiments, the method is used to identify and
study the organisms present in a human or animal or plant
"microbiome", e.g., for research, medical, or agricultural
applications. In some embodiments, the isolate rRNA sample is from
an initial sample comprising a sample from a human, animal, or
plant specimen that is provided for medical diagnostic or
theranostic testing or analysis (e.g., to detect a pathogenic
microorganism, such as a fungal or bacterial pathogen, that is or
may be related to or causative of a disease condition, e.g., a
pathogenic bacterium that can be detected or diagnosed based on
analysis of at least one rRNA molecule, e.g., as described by
Chakravorty, S et al., J. Microbiol. Methods 69: 330-339, 2007 and
by Millar, B C et al. in Current Issues Mol. Biol. 9: 21-40, 2007,
both incorporated herein by reference.) In some embodiments, the
isolated rRNA sample is analyzed using any of the many
next-generation sequencing methods known in the art (e.g., after
tagging the 3' end or the 3' and 5' ends of the isolated rRNA
molecules and reverse transcribing them to make tagged or di-tagged
linear ssDNA templates or circular ssDNA templates for
next-generation sequencing (e.g., using a Roche 454.TM., Illumina
Solexa.TM., Life Technologies SOLID.TM., Pacific Biosciences,
Intelligent Biosystems, Helicos, Qiagen, or another next-generation
sequencing platform. In still other embodiments, the isolated rRNA
sample is analyzed by real-time PCR.
[0038] In some embodiments, the present invention provides methods
for isolating substantially all of the RNA molecules that, either
alone or in combination, exhibit the sequence within at least one
full-length rRNA molecule, the method comprising: a) providing i)
an initial sample comprising RNA molecules, wherein the RNA
molecules comprise rRNA molecules and non-rRNA RNA molecules; and
ii) a composition comprising affinity-tagged antisense rRNA
molecules that are bound to a binding matrix (e.g., microparticles
comprising affinity-tag-binding molecules), wherein the
affinity-tagged antisense rRNA molecules, alone or in combination,
exhibit sequences corresponding to substantially all of at least
one full-length rRNA molecule selected from: 25S, 26S, 28S, 18S,
5.8S, and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules; b) contacting the initial sample with
the composition under conditions such that at least some of the
affinity-tagged antisense rRNA molecules in the composition and at
least some of the rRNA molecules form double-stranded rRNA hybrids
that are bound to the binding matrix, thereby generating a treated
sample; and c) removing the binding matrix to which the
double-strand rRNA hybrids are bound, thereby isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit the sequence within at least one full-length
rRNA molecule. In some embodiments of this method, the method
further comprises the step of washing the binding matrix to which
the double-stranded rRNA hybrids are bound in order to remove
non-specifically-bound non-rRNA RNA molecules from the sample. In
some embodiments, the treated sample is stored on the binding
matrix for future analysis or use. In some embodiments, the method
further comprises the step of incubating the binding matrix in a
solution under conditions wherein the at least some rRNA molecules
from the treated sample are released into the solution, thereby
generating a solution of the isolated rRNA sample comprising
substantially all of the RNA molecules that, either alone or in
combination, exhibit a sequence within at least one full-length
rRNA molecule present in the initial sample (e.g., at least >90%
. . . , >95% . . . , >98% . . . , >99% . . . , >99.8% .
. . , or >99.9% of the RNA molecules that, either alone or in
combination, exhibit a sequence within at least one full-length
rRNA molecule present in the initial sample) and wherein, either i)
the isolated rRNA sample is substantially free (e.g., at least
>90% . . . , >95% . . . , >98% . . . , >99% . . . ,
>99.8% . . . , or >99.9% free) of the non-rRNA RNA molecules
present in the initial sample, or ii) contains undetectable levels
of non-rRNA RNA from the initial sample as measured using agarose
gel electrophoresis and ethidium bromide staining. In some
embodiments, the isolated rRNA sample is from an environmental or
metagenomic sample and the isolated rRNA is analyzed to determine
the genera, species or strains that were present in the sample, and
therefore, the particular environment from which the sample was
collected. In some embodiments, the isolated rRNA sample is
analyzed (e.g., by next-generation sequencing, real-time reverse
transcription PCR, or another method, such as a method described
elsewhere herein).
[0039] In some embodiments of any of the methods of the invention
for generating a rRNA-depleted sample or for isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit the sequence of at least one full-length rRNA
molecule, the method uses any of the compositions comprising
affinity-tagged antisense rRNA molecules obtained using any of the
methods described herein for generating affinity-tagged antisense
rRNA molecules.
[0040] In some embodiments of any of the methods for generating a
rRNA-depleted sample or for isolating substantially all of the rRNA
that, either alone or in combination, exhibits a sequence within at
least one full-length rRNA molecule from an initial sample, the
composition comprising affinity-tagged antisense rRNA molecules
that are bound to a binding matrix comprises biotin as the affinity
tag, wherein the biotin is joined to at least about two to eight
nucleobases per hundred nucleobases of the antisense rRNA
molecules, and the binding matrix comprises microparticles to which
streptavidin or avidin is attached as the affinity-tag-binding
molecule. In some other embodiments of this method, the
affinity-tagged antisense rRNA molecules comprise biotin as the
affinity tag, wherein the biotin is joined to at least about two to
four nucleobases per hundred nucleobases of the antisense rRNA
molecules, or to at least about three to five nucleobases per
hundred nucleobases of the antisense rRNA molecules, or to at least
about four to six nucleobases per hundred nucleobases of the
antisense rRNA molecules, or to at least about six to eight
nucleobases per hundred nucleobases of the antisense rRNA
molecules.
[0041] In some embodiments of any of the methods of the invention
for generating a rRNA-depleted sample or for isolating
substantially all of the rRNA that, either alone or in combination,
exhibits a sequence within at least one full-length rRNA molecule
from an initial sample, the initial sample contains degraded RNA
and the method is used for generating a rRNA-depleted sample for
further analysis and use. For example, in some embodiments, the
initial sample is an FFPE sample that contains degraded RNA, and
the method is used for generating a rRNA-depleted sample that
contains at least one non-rRNA RNA molecule of interest, wherein
the rRNA-depleted sample is used for analysis of expression or
relative expression of one or more RNA molecules, selected from
among mRNA, miRNA, ncRNA, piwiRNA, and RNA comprising the whole
transcriptome (minus the rRNA) from one or multiple cells, tissues,
organs, or organisms, wherein the method of analysis is selected
from among microarray analysis (e.g., Affymetrix, NimbleGen
Systems, Agilent Systems), next-generation sequencing (or so-called
"digital" analysis), including, among others, next-gen RNA
sequencing methods and techniques referred to by terms such as
"RNA-Seq," "digital mRNA profiling," "transcriptome profiling," and
"ribosome profiling" (e.g., Ingolia et al., Science 324: 218-223,
2009), screening analysis, and analysis using a diagnostic or
theranostic assay, including a diagnostic or theranostic assay
comprising reverse-transcription qPCR, and a diagnostic or
theranostic assay comprising RNA amplification and/or detection of
a sequencing using a labeled probe.
[0042] In certain embodiments of any of the methods of the
invention for using a composition comprising affinity-tagged
antisense rRNA molecules for generating a rRNA-depleted sample or
for isolating substantially all of the RNA molecules that, either
alone or in combination, exhibit the sequence of at least one
full-length rRNA molecule, the composition of affinity-tagged
antisense rRNA molecules are generated from a first species or
organism, and used for generating a rRNA-depleted sample or for
isolating substantially all of the RNA molecules that, either alone
or in combination, exhibit a sequence within at least one
full-length rRNA molecule from a second species or organism
different from the first species or organism (e.g., the first
species or organism is mouse or rat and the second species or
organism is human). In some embodiments, the affinity-tagged
antisense rRNA molecules are generated from any at least one rRNA
molecule from the first species of organism wherein said
affinity-tagged antisense rRNA molecules hybridize with sequences
exhibited within all of the at least one rRNA molecules present in
the initial sample from the second species or organism under the
conditions used in the method for generating a rRNA-depleted sample
or for isolating at least one rRNA molecule from the initial
sample. In further embodiments, the first species or organism is a
non-human mammal (e.g., cat, dog, sheep, mouse, rat, monkey, etc.)
and the second species or organism is homo sapiens. In particular
embodiments, the first species or organism comprises non-E. coli
bacteria, and the second species or organism is E. coli. In some
embodiments, the method for generating a rRNA-depleted sample or
for isolating substantially all of the RNA molecules that, either
alone or in combination, exhibit a sequence within at least one
full-length rRNA molecule is performed using a metagenomic or
environmental sample containing multiple species or organisms.
Thus, in some embodiments wherein the composition comprising
affinity-tagged antisense rRNA molecules corresponds to at least
one full-length prokaryotic rRNA molecule (e.g., generated from at
least one full-length rRNA molecule from E. coli), the composition
is used for generating a rRNA-depleted sample or for isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit a sequence within at least one full-length
rRNA molecule comprising multiple rRNA molecules from multiple
species or organisms.
[0043] In some embodiments of any of the methods of the invention
for generating a rRNA-depleted sample or for isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit the sequence of at least one full-length rRNA
molecule, instead of using the compositions comprising
affinity-tagged antisense rRNA molecules, the method uses
compositions comprising affinity-tagged single-stranded DNA
molecules that, alone or in combination, are complementary to
substantially all of the sequence exhibited by the at least one
full-length rRNA molecule selected from: 25S, 26S, 28S, 18S, 5.8S,
and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules, wherein the affinity tags are present
at a ratio of at least about two to at least four affinity tags per
hundred nucleobases of the affinity-tagged single-stranded DNA
molecules.
[0044] Word conducted during developments of the present invention
found that the method can be performed using samples wherein the
initial sample comprises generally any amount of total RNA
molecules. In one embodiment, the amount of total RNA present in
the initial sample is between about 10 picograms and 50 nanograms.
In another embodiment, the amount of total RNA present in the
initial sample is between about 50 nanograms and one microgram. In
still another embodiment, the amount of total RNA present in the
initial sample is between about one microgram and five micrograms.
In some embodiments of these methods, at least a portion of the RNA
molecules in the initial sample are highly fragmented (e.g.,
previously digested with an RNase enzyme or from older RNA
samples). In some embodiments of these methods, the RNA molecules
in the initial sample are from a paraffin-embedded sample (e.g.,
paraffin-embedded formalin-fixed sample). In further embodiments of
these methods, the affinity-tagged antisense RNA molecules
corresponds to the at least one rRNA molecules from the same
species or organism (e.g., the affinity-tagged antisense RNA
molecules correspond to the at least one rRNA molecule from human
cells or the affinity-tagged antisense RNA molecules correspond to
the at least one rRNA molecule from E. coli cells). In certain
embodiments of these methods wherein the affinity-tagged antisense
RNA molecules corresponds to the at least one rRNA molecule from
human cells, the at least one rRNA molecule is human 28S rRNA.
[0045] In some embodiments of any of the methods of the invention
for generating a rRNA-depleted sample or for isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit the sequence of at least one full-length rRNA
molecule, the ratio of the affinity-tag-binding molecules to the
affinity tags during the contacting in step b) is at least 2 to 1
(e.g., 2:1, 3:1, 4:1, 5:1, . . . or 10:1).
[0046] In certain embodiments of any of the methods of the
invention for generating a rRNA-depleted sample or for isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit the sequence of at least one full-length rRNA
molecule, the initial sample comprising RNA molecules represents
total RNA isolated from a cell or tissue sample or from an
environmental sample (e.g., from a cell line, a tissue biopsy
sample, a bodily fluid sample, a swab from a hospital surface, a
water sample, etc.). In some embodiments of these methods, the
method further comprises providing a control sample of RNA (e.g.,
comprising total RNA) from a cell or tissue sample, and also
performing the method using the control sample. In certain
embodiments, the control sample comprises total RNA from HeLa cells
and/or from E. coli cells.
[0047] In additional embodiments of any of the methods of the
invention for generating a rRNA-depleted sample or for isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit the sequence of at least one full-length rRNA
molecule, the initial sample is substantially free of salts and/or
organic liquids. In other of these embodiments, the initial sample
comprises RNase-free water and/or TE buffer. In particular
embodiments, the method further comprises treating the
rRNA-depleted sample or the isolated rRNA sample under conditions
such that the RNA molecules present in the sample are further
purified (e.g., by further purification using ethanol, isopropanol
or ammonium acetate precipitation). In particular embodiments, the
rRNA-depleted sample or the isolated rRNA sample is used in methods
for further analysis, such as for analysis by next-gen sequencing,
for preparing labeled target for microarray analysis, for screening
or diagnostic or theranostic analysis (e.g., using a plant, human,
or animal, screening, diagnostic or theranostic kit). In some
embodiments, the nucleic acids in the rRNA-depleted sample or the
isolate rRNA sample is amplified prior to further analysis. In
other embodiments of these methods, the contacting in step b) is
conducted in the presence of an RNase inhibitor. In some
embodiments, the compositions used in the method further comprise
RNase free water.
[0048] In further embodiments of any of the methods of the
invention for generating a rRNA-depleted sample or for isolating
substantially all of the RNA molecules that, either alone or in
combination, exhibit the sequence of at least one full-length rRNA
molecule, the condition in step b) comprise incubating at a
temperate of about 60-75.degree. C. for a first time period (e.g.,
5-15 minutes) and incubating at about room temperature for a second
time period (e.g., 10-25 minutes). In some embodiments of these
methods, the conditions in step b) include the presence of
hybridization buffer. In particular embodiments of these methods,
the conditions in step c) include at least occasional mixing at
room temperature and/or at 35-60.degree. C. or 25-70.degree. C., or
at about 50.degree. C. In further embodiments of these methods, the
binding matrix comprises a plurality of individual particles (e.g.,
nanoparticle, magnetic particles, macroporous beads, etc.) In
further embodiments of these methods, the binding matrix comprises
a binding column or a membrane. In some embodiments of these
methods, the affinity tags are selected from biotin, avidin or
streptavidin, digoxigenin, antibodies (or antibody fragments), and
other useful binding molecules. In certain embodiments of these
methods, the affinity-tag-binding molecules are selected from
biotin, avidin or streptavidin, digoxigenin, antibodies (or
antibody fragments), and other useful binding molecules.
[0049] In some embodiments of the invention, one or more of the
sequences in the at least one rRNA molecules, or in one or more of
the non-rRNA RNA molecules, or in one or more other nucleic acid
molecules present in the initial sample is amplified (e.g., using
any appropriate method known in the art for RNA and/or DNA
amplification, e.g., real-time PCR or reverse transcription-PCR,
transcription-mediated amplification, RNA amplification using a
RiboMultiplier.TM. Kit, Epicentre Biotechnologies, Madison, Wis.,
strand-displacement amplification, LAMP, ICAN.TM., UCAN.TM.
(TAKARA), rolling circle amplification, etc.) either, prior to, or
after generating the rRNA-depleted sample or isolating the at least
one rRNA molecule, respectively, using one of the methods of the
present invention for generating a rRNA-depleted sample or for
isolating substantially all of the RNA molecules that, either alone
or in combination, exhibit the sequence within at least one
full-length rRNA molecule. In some of these embodiments, the sample
comprising the respective one or more amplified sequences of
non-rRNA RNA molecules, or rRNA molecules, and/or other nucleic
acid molecules is used for further analysis.
[0050] In other embodiments, the present invention provides a
composition comprising antisense rRNA molecules corresponding to
substantially all of at least one rRNA molecule selected from: 25S,
26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules,
12S and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S,
and 5S prokaryotic rRNA molecules, wherein the antisense rRNA
molecules comprise affinity-tags, wherein the composition is
substantially free of non-rRNA RNA molecules comprising the
affinity tags. In certain embodiments, the composition further
comprises a binding matrix, wherein the binding matrix comprises
affinity-tag-binding molecules (e.g., microparticles). In
particular embodiments, the antisense rRNA molecules are bound to
the binding matrix via the affinity-tag-affinity tag binding
molecule interaction.
[0051] In some embodiments, the present invention provides
compositions comprising: a) a composition comprising antisense rRNA
molecules corresponding to substantially all of at least one rRNA
molecule selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules,
wherein the antisense rRNA molecules comprise affinity tags, and b)
non-rRNA RNA molecules that are affinity-tag free. In certain
embodiments, the compositions further comprise a binding matrix
comprising affinity-tag-binding molecules.
[0052] In particular embodiments, the present invention provides
compositions comprising an rRNA-depleted sample comprising non-rRNA
RNA molecules, wherein the composition is substantially free of
rRNA sequences exhibited by at least one rRNA molecule selected
from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA
molecules, 12S and 16S eukaryotic mitochondrial rRNA molecules, and
23S, 16S, and 5S prokaryotic rRNA molecules.
[0053] In further embodiments, the present invention provides
compositions comprising antisense rRNA molecules corresponding to
substantially all of at least one rRNA molecule selected from: 25S,
26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules,
12S and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S,
and 5S prokaryotic rRNA molecules, wherein the antisense rRNA
molecules comprise affinity-tags, and wherein the composition is
substantially free of non-rRNA RNA molecules comprising the
affinity tags.
[0054] In other embodiments, the present invention provides
compositions comprising: a) compositions comprising antisense rRNA
molecules corresponding to substantially all of at least one rRNA
molecule selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules,
wherein the antisense rRNA molecules comprise affinity-tags, and b)
non-rRNA RNA molecules that are affinity-tag free. In further
embodiments, the compositions further comprise a binding matrix
comprising affinity-tag-binding molecules.
[0055] In some embodiments, the present invention provides
compositions comprising: an rRNA-depleted sample comprising
non-rRNA RNA molecules, wherein the composition is substantially
free of rRNA sequences exhibited by at least one rRNA molecule
selected from among 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules. In
some particular embodiments, the compositions are substantially
free of rRNA sequences exhibited by two, three, or four rRNA
molecules selected from among 25S, 26S, 28S, 18S, 5.8S, and 5S
eukaryotic cytoplasmic rRNA molecules. In some particular
embodiments, the compositions are substantially free of rRNA
sequences exhibited by both 12S and 16S eukaryotic mitochondrial
rRNA molecules.
[0056] In certain embodiments, the present invention provides
compositions comprising: a) compositions comprising antisense rRNA
molecules corresponding to substantially all of at least one rRNA
molecule selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules,
wherein the antisense rRNA molecules comprise affinity-tags, and
wherein the antisense rRNA molecules are from a first species or
organism, and b) non-rRNA RNA molecules that are affinity-tag free,
wherein the non-rRNA RNA molecules are from a second species or
organism different from the first species or organism. In other
embodiments, the compositions further comprise a binding matrix
comprising affinity-tag-binding molecules.
[0057] In some embodiments, the present invention provides
compositions comprising: a) affinity-tagged antisense rRNA
molecules corresponding to substantially all of at least one rRNA
molecule selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules,
wherein the affinity tags are present on the antisense rRNA
molecules at a ratio of at least about two to eight affinity tags
per hundred nucleobases of the antisense rRNA molecules, and b)
non-rRNA RNA molecules that are affinity-tag free. In some
embodiments, composition comprises affinity-tagged antisense rRNA
molecules from a first species or organism and non-rRNA RNA
molecules that are affinity-tag free from a second species or
organism. In certain embodiments, the compositions further comprise
a binding matrix comprising affinity-tag-binding molecules.
[0058] In additional embodiments, the present invention provides
compositions comprising: an rRNA-depleted sample comprising
non-rRNA RNA molecules, wherein the composition is at least about
99.0% free of rRNA sequences exhibited by at least one rRNA
molecule selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA
molecules.
[0059] The present invention provides kits or systems for
performing any of the methods of the invention, including any of
the steps of said methods.
[0060] For example, in some further embodiments, the present
invention provides kits and systems comprising: a) a first
component comprising a composition comprising antisense rRNA
molecules complementary to substantially all of the sequence
exhibited by the at least one rRNA molecule selected from: 25S,
26S, 28S, 18S, 5.8S, and 5S eukaryotic cytoplasmic rRNA molecules,
12S and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S,
and 5S prokaryotic rRNA molecules, wherein the antisense rRNA
molecules comprise affinity-tags, and wherein the composition is
substantially free of non-rRNA RNA molecules comprising the
affinity tags; and b) at least one second component selected from
the group consisting of: i) a binding matrix comprising
affinity-tag-binding molecules; ii) a control sample comprising
total RNA from a cell or tissue sample; iii) a solution comprising
an RNase inhibitor; iv) a binding matrix wash solution that is
RNase-free; v) a volume of RNase-free water; vi) a hybridization
buffer; vii) a total RNA purification reagent; and viii) a binding
matrix resuspension solution, wherein said solution is RNase-free.
In certain embodiments, the second component is the binding matrix.
In further embodiments, the second component is the control sample.
In some embodiments, the second component is the solution
comprising an RNase inhibitor.
[0061] In some embodiments, the present invention provides kits and
systems comprising: a) a first component comprising a composition
comprising antisense rRNA molecules complementary to substantially
all of the sequence exhibited by the at least one rRNA molecule
selected from: 25S, 26S, 28S, 18S, 5.8S, and 5S eukaryotic
cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial
rRNA molecules, and 23S, 16S, and 5S prokaryotic rRNA molecules,
wherein the antisense rRNA molecules comprise affinity-tags, and
wherein the composition is substantially free of non-rRNA RNA
molecules comprising the affinity tags; and b) at least one second
component selected from the group consisting of: i) a binding
matrix comprising affinity-tag-binding molecules; ii) a control
sample comprising total RNA from a cell or tissue sample; iii) a
solution comprising an RNase inhibitor; iv) a binding matrix wash
solution that is RNase-free; v) a volume of RNase-free water; vi) a
hybridization buffer; and vii) a total RNA purification
reagent.
[0062] In additional embodiments, the present invention provides
kits and systems comprising: a) a first component comprising a
composition comprising affinity-tagged antisense rRNA molecules
complementary to substantially all of the sequence exhibited by the
at least one rRNA molecule selected from: 25S, 26S, 28S, 18S, 5.8S,
and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S
eukaryotic mitochondrial rRNA molecules, and 23S, 16S, and 5S
prokaryotic rRNA molecules, wherein the antisense rRNA molecules
comprise affinity tags, wherein the affinity tags are present on
the antisense rRNA molecules at a ratio of at least about two to
eight affinity tags per hundred nucleobases of the antisense rRNA
molecules, and wherein the composition is substantially free of
non-rRNA RNA molecules comprising the affinity tags; and b) at
least one second component selected from the group consisting of:
i) a binding matrix comprising affinity-tag-binding molecules; ii)
a control sample comprising total RNA from a cell or tissue sample;
iii) a solution comprising an RNase inhibitor; iv) a binding matrix
wash solution that is RNase-free; v) a volume of RNase-free water;
vi) a hybridization buffer; and vii) a total RNA purification
reagent.
[0063] In some embodiments of a kit or system, the affinity-tags
are present in the composition of affinity-tagged antisense rRNA
molecules at a ratio of at least about two to about eight affinity
tags per every 100 nucleobases of the antisense rRNA molecules
(e.g., at least 2:100, 3:100, 4:100, 5:100, 6:100, 7:100, or
8:100). In certain embodiments, the affinity tag is associated with
only one nucleobase of the antisense rRNA molecules. In some of
these embodiments, the affinity-tags are associated with only one
type of nucleobase selected from: adenine (A), cytosine (C),
guanine (G) and uracil (U).
[0064] In other embodiments of a kit or system, the compositions
comprising affinity-tagged antisense rRNA molecules correspond to
at least 95.0% of all of the at least one rRNA sequence (e.g.,
95.0% . . . 95.5% . . . 96.0% . . . 96.5% . . . 97.0% . . . 97.5% .
. . 98% . . . 98.5% . . . 99.0% . . . 99.5% . . . 99.9 . . . or
100% of the at least one rRNA molecule (e.g., wherein the
compositions comprise antisense rRNA molecules that exhibit, alone
or in combination, sequences corresponding to all of the
full-length rRNA sequence for a particular rRNA molecule).
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] The foregoing summary and description is better understood
when read in conjunction with the accompanying drawings which are
included by way of example and not by way of limitation.
[0066] FIG. 1A shows an ethidium bromide stained agarose gel
containing the following PCR amplicons: Lane M--DNA molecular
weight ladder; Lane 1--300 ng of PCR amplicon for human 18S rRNA;
Lane 2--300 ng of PCR amplicon for human 5.8S rRNA; Lane 3--300 ng
of PCR amplicon for human 5S rRNA; Lane 4--300 ng of PCR amplicon
for E. coli 23S rRNA 5' segment; Lane 5--300 ng of PCR amplicon for
E. coli 23S rRNA 3' segment; Lane 6--300 ng of PCR amplicon for E.
coli 16S rRNA; and Lane 7--300 ng of PCR amplicon for E. coli 5SS
rRNA
[0067] FIG. 1B shows an ethidium bromide stained agarose gel
containing the following antisense sequences: Lane M--DNA molecular
weight ladder; Lane 1--300 ng of human antisense 18S rRNA; Lane
2--300 ng of human antisense 5.8S rRNA; Lane 3--300 ng of human
antisense 5S rRNA; Lane 4--300 ng of E. coli antisense 23S rRNA 5'
segment; Lane 5--300 ng of E. coli antisense 23S rRNA 3' segment;
Lane 6--300 ng of E. coli antisense 16S rRNA; and Lane 7--300 ng of
E. coli antisense 5S rRNA.
[0068] FIG. 1C shows an ethidium bromide stained agarose gel
containing the following lanes: Lane M--DNA molecular weight
ladder; Lane 1--300 ng of T7-based 28S biotinylated antisense rRNA;
and Lane 2--300 ng of SP6-based 28S biotinylated antisense
rRNA.
[0069] FIG. 2A shows the following: Lane M--DNA molecular weight
ladder; Lane 1--Biotinylated antisense rRNA following purification
with microspheres directly on column; Lane 2--Biotinylated
antisense rRNA following purification from supernatant after
removal of microspheres; and Lane 3--Biotinylated antisense rRNA
control.
[0070] FIG. 2B shows the following: Lane M--DNA molecular weight
ladder; Lane 1--Biotinylated antisense rRNA control; Lane
2--Biotinylated antisense rRNA following purification after 20
.mu.l of microspheres; Lane 3--Biotinylated antisense rRNA
following purification after 2.times.20 .mu.l of microspheres; and
Lane 4--Biotinylated antisense rRNA following purification after 40
.mu.l of microspheres
[0071] FIG. 3A shows the following: Lane M--DNA molecular weight
ladder; Lane 1 --10% biotinylated antisense rRNA minus
microspheres; Lane 2--10% biotinylated antisense rRNA plus 20 .mu.l
microspheres; Lane 3--10% biotinylated antisense rRNA plus 40 .mu.l
microspheres; Lane 4--20% biotinylated antisense rRNA minus
microspheres; Lane 5--20% biotinylated antisense rRNA plus 20 .mu.l
microspheres; and Lane 6--20% biotinylated antisense rRNA plus 40
.mu.l microspheres.
[0072] FIG. 3B shows the following: Lane M--DNA molecular weight
ladder; Lane 1--35% biotinylated antisense rRNA minus streptavidin
microspheres; Lane 2--35% biotinylated antisense rRNA plus
streptavidin microspheres; Lane 3--50% biotinylated antisense rRNA
minus streptavidin microspheres; Lane 4--50% biotinylated antisense
rRNA plus streptavidin microspheres; Lane 5--60% biotinylated
antisense rRNA minus streptavidin microspheres; and Lane 6--60%
biotinylated antisense rRNA plus streptavidin microspheres.
[0073] FIG. 3C shows the following: Lane M--DNA molecular weight
ladder; Lane 1--PCR of 35% biotinylated antisense rRNA minus
streptavidin microspheres; Lane 2--PCR of 35% biotinylated
antisense rRNA plus streptavidin microspheres; Lane 3--PCR of 50%
biotinylated antisense rRNA minus streptavidin microspheres; Lane
4--PCR of 50% biotinylated antisense rRNA plus streptavidin
microspheres; Lane 5--PCR of 60% biotinylated antisense rRNA minus
streptavidin microspheres; and Lane 6--PCR of 60% biotinylated
antisense rRNA plus streptavidin microspheres. Panel 1 shows 5'-23S
rRNA primers. Panel 2 shows 3'-23S rRNA primers. Panel 3 shows
5'-16S rRNA primers. Panel 4 shows 3'-16S rRNA primers.
[0074] FIG. 4 shows the following: Lane M--DNA molecular weight
ladder; Lane 1--L. plantarum total RNA plus E. coli biotinylated
antisense rRNA mixture subtraction; and Lane 2--L. plantarum total
RNA minus E. coli biotinylated antisense rRNA mixture
subtraction.
[0075] FIG. 5, panels A-D, showing the following: Lane M--DNA
molecular weight ladder; Lanes 1, 2--PCR result for 50 ng input E.
coli total RNA plus subtraction; Lanes 3,4--PCR result for 100 ng
input E. coli total RNA plus subtraction; Lanes 5, 6--PCR result
for 500 ng input E. coli total RNA plus subtraction; Lanes 7,8--PCR
result for 1000 ng input E. coli total RNA plus subtraction; Lanes
9,10--PCR result for 2500 ng input E. coli total RNA plus
subtraction; Lanes 11,12--PCR result for 5000 ng input E. coli
total RNA plus subtraction; Lanes 13,14--PCR result for 500 ng
input E. coli total RNA minus subtraction; and Lane 15--PCR result
for no template control reaction. Panel 5A shows 5'-23S rRNA
RT-PCR. Panel 5B shows 5'-16S rRNA RT-PCR. Panel 5C shows 5S rRNA
RT-PCR. Panel 5D shows ompA mRNA RT-PCR.
[0076] FIG. 6A shows the following: Lane M--DNA molecular weight
ladder; Lane 1 --300 ng intact E. coli total RNA; and Lane 2--300
ng fragmented E. coli total RNA.
[0077] FIG. 6B shows the following: Lane M--DNA molecular weight
ladder; Lane 1 --Intact E. coli total RNA minus subtraction; Lane
2--Intact E. coli total RNA plus subtraction; Lane 3--Fragmented E.
coli total RNA minus subtraction; and Lane 4--Fragmented E. coli
total RNA plus subtraction. Panel 1 shows an ethidium bromide
stained agarose gel. Panel 2 shows ompA mRNA Northern blot. Panel 3
shows 23S rRNA Northern blot. Panel 4 shows 16S rRNA Northern blot.
Panel 5 shows 5S rRNA Northern blot.
[0078] FIG. 6C shows the following: Lane M--DNA molecular weight
ladder; Lane 1 --Intact E. coli total RNA minus subtraction; Lane
2--Intact E. coli total RNA plus subtraction; Lane 3--Fragmented E.
coli total RNA minus subtraction; and Lane 4--Fragmented E. coli
total RNA plus subtraction. Panel 1 shows ompA RT-PCR. Panel 2
shows 23S rRNA 5' RT-PCR. Panel 3 shows 16S rRNA 5' RT-PCR. Panel 4
shows 5S rRNA RT-PCR.
[0079] FIG. 7A shows the following: Lane M--DNA molecular weight
ladder; Lane 1--biotinylated antisense 28S rRNA; Lane
2--biotinylated antisense 18S rRNA; Lane 3--Human total RNA; Lane
4--Human total RNA plus biotinylated antisense 28S rRNA; Lane
5--Human total RNA plus biotinylated antisense 18S rRNA; Lane
6--Mouse total RNA; Lane 7--Mouse total RNA plus biotinylated
antisense 28S rRNA; Lane 8--Mouse total RNA plus biotinylated
antisense 18S rRNA; Lane 9--Rat total RNA; Lane 10--Rat total RNA
plus biotinylated antisense 28S rRNA; and Lane 11--Rat total RNA
plus biotinylated antisense 18S rRNA.
[0080] FIG. 7B shows the following: Lane M--DNA molecular weight
ladder; Lane 1 --Human total RNA (HeLa) minus subtraction; Lane
2--Human total RNA (HeLa) plus subtraction; Lane 3--Mouse total RNA
(3T3) minus subtraction; Lane 4--Mouse total RNA (3T3) minus
subtraction; Lane 5--Rat total RNA (NRK) minus subtraction; and
Lane 6--Rat total RNA (NRK) plus subtraction.
[0081] FIG. 8 shows the following: Lane M--DNA molecular weight
ladder; Lane 1--PCR result for 100 ng input human total RNA plus
subtraction; Lane 2--PCR result for 500 ng input human total RNA
plus subtraction; Lane 3--PCR result for 5.0 .mu.g input human
total RNA plus subtraction; Lane 4--PCR result for 500 ng input
human total RNA minus subtraction; and Lane 5--PCR result for no
template control reaction. Panel A shows 5' 28S rRNA RT-PCR. Panel
B shows 3' 28S rRNA RT-PCR. Panel C shows 5' 18S rRNA RT-PCR. Panel
D shows 3' 18S rRNA RT-PCR. Panel E shows 5.8S rRNA RT-PCR. Panel F
shows 5S rRNA RT-PCR. Panel G shows 5' GAPDH mRNA RT-PCR.
[0082] FIG. 9A shows the following: Lane M--DNA molecular weight
ladder; Lane 1 --Intact Hela total RNA minus subtraction; Lane
2--Intact HeLa total RNA plus subtraction; Lane 3--Fragmented (1
minute) Hela total RNA minus subtraction; Lane 4--Fragmented (1
minute) Hela total RNA plus subtraction; Lane 5--Fragmented (2
minute) Hela total RNA minus subtraction; Lane 6--Fragmented (2
minute) Hela total RNA plus subtraction; Lane 7--Fragmented (3
minute) Hela total RNA minus subtraction; and Lane 8--Fragmented (3
minute) Hela total RNA plus subtraction.
[0083] FIG. 9B shows the following: Lane 1--PCR result for intact
HeLa total RNA minus subtraction; Lane 2--PCR result for intact
HeLa total RNA plus subtraction; Lane 3--PCR result for fragmented
(1 minute) HeLa total RNA minus subtraction; Lane 4--PCR result for
fragmented (1 minute) HeLa total RNA plus subtraction; Lane 5--PCR
result for fragmented (2 minute) HeLa total RNA minus subtraction;
Lane 6--PCR result for fragmented (2 minute) HeLa total RNA plus
subtraction; Lane 7--PCR result for fragmented (3 minute) HeLa
total RNA minus subtraction; Lane 8--PCR result for fragmented (3
minute) HeLa total RNA plus subtraction; and Lane 9--PCR result for
no template control reaction. Panel 1 shows 5' 28S rRNA. Panel 2
shows 5' 18S rRNA. Panel 3 shows 5.8S rRNA. Panel 4 shows 5S rRNA.
Panel 5 shows 5' GAPDH mRNA
[0084] FIG. 10A shows the following: Lane M--DNA molecular weight
ladder; Lane 1--Intact E. coli total RNA minus subtraction; Lane
2--Intact E. coli total RNA plus subtraction; Lane 3--Fragmented E.
coli total RNA minus subtraction; and Lane 4--Fragmented E. coli
total RNA plus subtraction. Panel 1 shows ompA mRNA. Panel 2 shows
23S rRNA. Panel 3 shows 16S rRNA. Panel 4 shows 5S rRNA.
[0085] FIG. 10B shows the following: Lane M--DNA molecular weight
ladder; Lane 1--PCR result for intact E. coli total RNA minus
subtraction; Lane 2--PCR result for intact E. coli total RNA plus
subtraction; Lane 3--PCR result for fragmented E. coli total RNA
minus subtraction; and Lane 4--PCR result for fragmented E. coli
total RNA plus subtraction. Panel 1 shows ompA mRNA. Panel 2 shows
23S rRNA. Panel 3 shows 16S rRNA. Panel 4 shows 5S rRNA.
[0086] FIG. 11A shows the following: Lane M--DNA molecular weight
ladder; Lane 1--Intact HeLa total RNA minus subtraction; Lane
2--Intact HeLa total RNA plus subtraction; Lane 3--Fragmented HeLa
total RNA minus subtraction; and Lane 4--Fragmented HeLa total RNA
plus subtraction. Panel 1 shows results of an exemplary method of
the present invention. Panel 2 shows the results of the OLIGOTEX
method.
[0087] FIG. 11B shows the following: Lane M--DNA molecular weight
ladder; Lane 1--PCR results for intact HeLa total RNA minus
subtraction; Lane 2--PCR results for intact HeLa total RNA plus
subtraction; Lane 3--PCR results for fragmented HeLa total RNA
minus subtraction; and Lane 4--PCR results for fragmented HeLa
total RNA plus subtraction. Panel 1 shows 5'-28S rRNA. Panel 2
shows 3'-28S rRNA. Panel 3 shows 5'-18S rRNA. Panel 4 shows 3'-18S
rRNA. Panel 5 shows 5.8S rRNA. Panel 6 shows 5S rRNA.
[0088] FIG. 11C shows the following: Lane M--DNA molecular weight
ladder; Lane 1--PCR results for intact HeLa total RNA minus
subtraction; Lane 2--PCR results for intact HeLa total RNA plus
subtraction; Lane 3--PCR results for fragmented HeLa total RNA
minus subtraction; and Lane 4--PCR results for fragmented HeLa
total RNA plus subtraction. Panel 1 shows 5'-GAPDH. Panel 2 shows
3'-GAPDH. Panel 3 shows 5'-PGK1. Panel 4 shows 3'-PGK1.
[0089] FIG. 11D shows the following: Lane M--DNA molecular weight
ladder; Lane 1--PCR results for intact HeLa total RNA minus
subtraction; Lane 2--PCR results for intact HeLa total RNA plus
subtraction; Lane 3--PCR results for fragmented HeLa total RNA
minus subtraction; and Lane 4--PCR results for fragmented HeLa
total RNA plus subtraction. Panel 1 shows Poly A- RNA #1. Panel 2
shows Poly A- RNA #3. Panel 3 shows Poly A- RNA #15. Panel 4 shows
Bimorphic RNA #5.
[0090] FIG. 12A shows the following: Lane M--DNA molecular weight
ladder; Lane 1--Intact HeLa total RNA minus subtraction; Lane
2--Intact HeLa total RNA plus subtraction; Lane 3--Fragmented HeLa
total RNA minus subtraction; and Lane 4--Fragmented HeLa total RNA
plus subtraction.
[0091] FIG. 12B shows Lane M--DNA molecular weight ladder; Lane
1--PCR results for intact HeLa total RNA minus subtraction; Lane
2--PCR results for intact HeLa total RNA plus subtraction; Lane
3--PCR results for fragmented HeLa total RNA minus subtraction; and
Lane 4--PCR results for fragmented HeLa total RNA plus subtraction;
Panel 1--5'-28S rRNA; Panel 2--3'-28S rRNA; Panel 3--5'-18S rRNA;
Panel 4--3'-18S rRNA; Panel 5--5.8S rRNA; and Panel 6--5S rRNA.
[0092] FIG. 12C shows Lane M--DNA molecular weight ladder; Lane
1--PCR results for intact HeLa total RNA minus subtraction; Lane
2--PCR results for intact HeLa total RNA plus subtraction; Lane
3--PCR results for fragmented HeLa total RNA minus subtraction;
Lane 4--PCR results for fragmented HeLa total RNA plus subtraction;
Panel 1--5'-GAPDH; and Panel 2--3'-GAPDH.
DEFINITIONS
[0093] It is to be understood that the terminology used herein is
for the purpose of describing particular embodiments only, and is
not intended to be limiting. Further, unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention pertains. In describing and claiming the present
invention, the following terminology and grammatical variants will
be used in accordance with the definitions set forth below.
[0094] When the terms "for example", "e.g.", "such as", "include",
"including" or variations thereof are used herein, these terms will
not be deemed to be terms of limitation, and will be interpreted to
mean "but not limited to" or "without limitation."
[0095] As used herein, a "composition comprising affinity-tagged
antisense rRNA molecules" means "a composition comprising RNA
molecules that, either alone or in combination, exhibit one or more
sequences that are complementary to substantially all of the
sequence exhibited by the at least one full-length rRNA molecule,
wherein at least a portion of the nucleotides in said RNA molecules
are joined to an affinity tag."
[0096] As used herein, the phrase "a composition comprising
antisense rRNA molecules corresponding to substantially all of at
least one rRNA molecule" or "a composition comprising antisense
rRNA molecules corresponding to substantially all of (or "all of
the sequence exhibited by") "at least one rRNA molecule" or "a
composition comprising antisense rRNA molecules that, alone or in
combination, are complementary to substantially all" (or "all of
the sequence") "of at least one full-length rRNA molecule" refers
to a composition comprising affinity-tagged antisense rRNA
molecules that exhibit, that will specifically hybridize with or
anneal with or complex with, at least 95% of the RNA molecules or
fragments of RNA molecules that exhibit a sequence of a particular
full-length rRNA molecule. Preferably, the antisense rRNA molecules
in the composition will specifically hybridize with at least 95% of
the molecules of a particular rRNA molecule or fragments thereof in
a sample, such that, when affinity-tagged, said antisense rRNA
molecules can be used with a binding matrix to remove at least
about 95% of the RNA molecules or fragments of RNA molecules that
exhibit a sequence of said particular rRNA molecule from the
sample. It is not necessary that the antisense nucleic acid
sequences exhibited in the composition share 95% sequence identity
with the complement of a particular rRNA molecule, but instead, it
is only necessary that the antisense nucleic acid sequences are
able to specifically hybridize, anneal or complex with at least 95%
of the RNA molecules or fragments of RNA molecules that exhibit a
sequence of said particular rRNA molecule. The antisense nucleic
acid sequences in the composition do not need to be present in a
single nucleic acid strand (although they may be), but instead, the
composition comprising antisense rRNA molecule can consist of
antisense rRNA fragments that will collectively hybridize with at
least 95% of the RNA molecules or fragments of RNA molecules that
exhibit a sequence of a particular rRNA molecule. In certain
embodiments, the antisense nucleic acid sequences will specifically
hybridize with at least 95% . . . 96% . . . 97% . . . 98% . . .
98.5% . . . 99.0% . . . 99.3% . . . 99.5% . . . 99.8%, 99.0% . . .
99.5% . . . 99.9 . . . or 100% of the RNA molecules or fragments of
RNA molecules that exhibit the sequence of a particular rRNA
molecule.
[0097] A used herein, the phrase "an rRNA-depleted sample that
comprises substantially all of said at least one non-rRNA RNA
molecule of interest present in said initial sample," refers to a
purified sample that originally contained rRNA molecules and a
first amount of the at least one non-rRNA RNA molecule of interest
and that has been purified by removing a certain amount of rRNA
molecules, while retaining at least 90% of said first amount of
said at least one non-rRNA RNA molecule of interest. In certain
embodiments, at least 91% . . . 93% . . . 96% . . . 97% . . . 98% .
. . 98.5% . . . 99.0% . . . 99.3% . . . 99.5% . . . 99.8% . . . ,
or 99.9% of said first amount of said at least one non-rRNA RNA
molecule of interest is present in the rRNA-depleted sample. Those
with knowledge in the art will know or easily find methods for
assaying for the first amount of the at least one non-rRNA RNA
molecule of interest in the initial sample and assaying for the
amount of the at least one non-rRNA RNA molecule of interest
remaining in the rRNA-depleted sample, and using said information
to calculate the percentage of said first amount of said at least
one non-rRNA RNA molecule of interest that is present in the
rRNA-depleted sample. For example, in one embodiment, the
percentage of the at least one non-rRNA RNA molecule of interest
remaining in the rRNA-depleted sample is determined based on the
amounts of the at least one non-rRNA RNA molecule of interest in
the initial sample and in the rRNA-depleted sample as determined
using reverse transcription real-time PCR (also called
"RT-qPCR").
[0098] A used herein, the phrase "an isolated rRNA sample that is
substantially free of the non-rRNA RNA molecules present in the
initial sample" refers to a purified sample that initially
contained non-rRNA RNA molecules and a first amount of the at least
one rRNA molecule and that has been purified by removing a certain
amount of the non-rRNA RNA molecules, while retaining at least 90%
of said first amount of said at least one rRNA molecule. In certain
embodiments, at least >90% . . . , >95% . . . , >98% . . .
, >99% . . . , >99.8% . . . , or >99.9% of said first
amount of said at least one rRNA molecule is present in the
isolated rRNA sample. In certain embodiments, at least >90% . .
. , >95% . . . , >98% . . . , >99% . . . , >99.8% . . .
, or >99.9% of the non-rRNA RNA molecules that were present in
the initial sample are not present in the isolated rRNA sample
(e.g., wherein the amounts of the at least one rRNA molecule and/or
of the non-rRNA molecules are determined using RT-qPCR as described
above).
[0099] As used herein, the phrase "substantially free of RNA
molecules that exhibit sequences of the at least one rRNA molecule"
refers to a purified sample wherein 5% or less of all the molecules
present in the initial sample that exhibited a nucleic acid
sequence from said at least one rRNA molecule are still present in
the rRNA-depleted sample. In certain embodiments, 5% . . . 4% . . .
3% . . . 2% . . . 1.5% . . . 1.0% . . . 0.5% . . . or 0.1% or less
of all the molecules present in the initial sample that exhibited a
nucleic acid sequence from a particular rRNA molecule are still
present in the purified sample. Those with knowledge in the art
will know or easily find methods for assaying for the amounts of
RNA molecules that exhibit sequences of the at least one rRNA
molecule that are present in the initial sample and in the
rRNA-depleted sample. For example, in one embodiment, the
percentage of RNA molecules that exhibit sequences of the at least
one rRNA molecules is determined based on the amounts of the RNA
molecules that exhibit sequences of the at least one rRNA molecule
in the initial sample and in the rRNA-depleted sample as determined
using reverse transcription real-time PCR (also called
"RT-qPCR").
[0100] As used herein, the phrase "substantially free of non-rRNA
RNA molecules comprising affinity tags," refers to a composition
wherein 2% or less of all the affinity-tagged nucleic acid
sequences present are non-rRNA RNA molecules having affinity tags.
In certain embodiments, 2% . . . 1.5% . . . 1.0% . . . 0.5% . . .
0.1% or less of all the affinity-tagged nucleic acid molecules
present are non-rRNA RNA molecules having affinity tags.
[0101] As used herein, the terms "an rRNA-depleted sample" or "a
sample that is substantially free of rRNA molecules" is sometimes
referred to herein as "a rRNA subtracted sample" or "a subtracted
sample," and the "methods for generating rRNA-depleted samples" or
the "methods for isolating rRNA from samples" are sometimes
referred to herein as "methods for rRNA subtraction" or, more
simply, as "rRNA subtraction." The term "rRNA subtraction" herein
shall mean and refer to "a method for generating a rRNA-depleted
sample or a method for isolating rRNA from a sample." Similarly,
when a form of the verb "to subtract" is used herein, it shall mean
"a method or the process of performing a method for generating a
rRNA-depleted samples or for isolating rRNA from a sample" and the
term "subtracted" herein shall mean or refer to the rRNA-depleted
state of the sample after performing the method or process.
[0102] As used herein, the phrase "binding matrix" refers to any
type of substrate, whether porous or non-porous, that will bind
affinity-tagged nucleic acid molecules such that affinity-tagged
nucleic acid molecules, and the sequences they are hybridized to,
can can be preferentially removed from a sample.
Affinity-tag-binding molecules are associated with the binding
matrix to allow the binding matrix to bind affinity-tagged nucleic
acid molecules. Examples of such binding matrices include, but are
not limited to, nylon membranes or particles, silica membranes or
particles, cellulose acetate membranes or particles, membranes or
particles composed of silica and Fe.sub.2O.sub.3, and other similar
membranes, fibers, coated plates, solid supports, and
particles.
[0103] As used herein, a "specific binding pair" refers to two
molecules that have affinity for and "bind" to each other under
certain conditions, referred to as "binding conditions." Biotin and
streptavidin or avidin are examples of a "specific binding pair" or
"affinity binding molecules," but the invention is not limited to
use of this particular specific binding pair. In many embodiments
of the present invention, one member of a particular specific
binding pair is referred to as the "affinity tag molecule" or the
"affinity tag" and the other as the "affinity-tag-binding molecule"
or the "affinity tag binding molecule." For example, but without
limitation, in some embodiments, biotin is referred to as the
affinity tag or affinity tag molecule, and a streptavidin or avidin
molecule, whether it is free, attached to a surface, attached to
another molecule, or labeled with a detectable molecule such as a
dye, is referred to as the affinity-tag-binding molecule. In other
embodiments, streptavidin is the affinity tag and biotin is the
affinity-tag-binding molecule, since streptavidin and biotin
function together as a specific binding pair or as affinity binding
molecules. A wide variety of other specific binding pairs or
affinity binding molecules, including both affinity tag molecules
and affinity-tag-binding molecules, are known in the art (e.g., see
U.S. Pat. No. 6,562,575, herein incorporated by reference), which
can be used in the present invention. For example, an antigen
(which itself may be an antibody) and an antibody, including a
monoclonal antibody, that binds the antigen is a specific binding
pair. Also, an antibody and an antibody binding protein, such as
Staphylococcus aureus Protein A, can be employed as a specific
binding pair. Other examples of specific binding pairs include, but
are not limited to, a carbohydrate moiety which is bound
specifically by a lectin and the lectin; a hormone and a receptor
for the hormone; and an enzyme and an inhibitor of the enzyme.
Usually, molecules that comprise a specific binding pair interact
with each other through non-covalent bonds such as
hydrogen-bonding, hydrophobic interactions (including stacking of
aromatic molecules), van der Waals forces, and salt bridges.
Without being bound by theory, it is believed in the art that these
kinds of non-covalent bonds result in binding, in part due to
complementary shapes or structures of the molecules involved in the
binding pair. The term "binding" according to the invention refers
to the interaction between an affinity binding molecules or
specific binding pairs (e.g., between biotin as an affinity tag
molecule and streptavidin as an affinity-tag-binding molecule) as a
result of non-covalent bonds, such as, but not limited to, hydrogen
bonds, hydrophobic interactions, van der Waals bonds, and ionic
bonds. Based on the definition for "binding," and the wide variety
of affinity binding molecules or specific binding pairs, it is
clear that "binding conditions" vary for different specific binding
pairs. Those skilled in the art can easily determine conditions
whereby, in a sample, binding occurs between the affinity binding
molecules. In particular, those skilled in the art can easily
determine conditions whereby binding between affinity binding
molecules that would be considered in the art to be "specific
binding" can be made to occur. As understood in the art, such
specificity is usually due to the higher affinity between the
affinity binding molecules than for other substances and components
(e.g., vessel walls, solid supports) in a sample. In certain cases,
the specificity might also involve, or might be due to, a
significantly more rapid association of affinity binding molecules
than with other substances and components in a sample.
[0104] In some embodiments of the invention, an "affinity tag
reagent" or and "affinity tag having a reactive moiety" is used, by
which we mean herein, a molecule that comprises both an affinity
tag and a reactive chemical group or moiety that is capable of
reacting with one or more atoms or groups of the molecule with
which it reacts to form one or more covalent chemical bonds between
the molecule comprising the affinity tag and the molecule with
which it reacts. By way of example, but without limitation, in some
embodiments, the affinity tag reagent is an acylating reagent
(e.g., an N-hydroxysuccinimidyl ester), wherein the affinity tag is
chemically joined to an atom in the molecule with which it reacts
via an acyl linkage. In other embodiments, the affinity tag reagent
is an alkylating reagent, group, wherein the affinity tag is
chemically joined to an atom in the molecule with which it reacts
via an alkyl linkage. In other embodiments, the affinity tag
reagent reacts via an electrocyclic type of chemical reaction, such
as a 1,3-dipolar cycloaddition (e.g., cycloaddition of an alkyne
with an azide). Thus, the term "reactive" moiety with respect to,
for example, an "affinity tag reagent" or "affinity tag having a
reactive moiety" is used to refer to a moiety or group that is
involved in or responsible for the chemical reaction whereby a
molecule comprising the affinity tag reacts chemically to form a
covalent chemical bond with one or more atoms in the molecule with
which it reacts, rather than to the binding that results between
affinity binding molecules due to non-covalent forces and
bonds.
[0105] When we refer to attaching the "affinity-tag-binding
molecule" or the "affinity tag binding molecule," such as
streptavidin or avidin, directly to the surface or solid support,
it usually, but not always means that the affinity-tag-binding
molecule is covalently attached to the surface by means of a
chemical linker that is joined to the surface and to the
affinity-tag-binding molecule. When we refer to attaching the
"affinity-tag-binding molecule" or the "affinity tag binding
molecule," such as streptavidin or avidin, indirectly to the
surface, it is meant that the affinity-tag-binding molecule is
bound to another molecule with which it has affinity (e.g., an
anti-streptavidin antibody) that is in turn bound to the surface.
In some embodiments, the affinity-tag-binding molecule, such as
streptavidin or avidin, is not attached to a surface, but is bound
by another molecule, such as an antibody or Protein A, and the
biotinylated modified-nucleotide-capped RNA is recovered by
precipitation or by binding to a second antibody or other molecule
using methods and compositions known in the art.
[0106] As used herein, the term "about" means encompassing plus or
minus 25%. For example, "about 200 nucleotides" refers to a range
encompassing between 150 and 250 nucleotides.
[0107] As used herein, the term "hybridization" or "hybridize" is
used in reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is influenced by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the melting temperature
(T.sub.m) of the formed hybrid, and the G:C ratio within the
nucleic acids. A single molecule that contains pairing of
complementary nucleic acids within its structure is said to be
"self-hybridized." An extensive guide to nucleic hybridization may
be found in Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology-Hybridization with Nucleic Acid Probes, part I,
chapter 2, "Overview of principles of hybridization and the
strategy of nucleic acid probe assays," Elsevier (1993), which is
incorporated by reference.
DETAILED DESCRIPTION
[0108] The present invention provides methods, compositions, and
kits for generating rRNA-depleted samples and for isolating rRNA
from samples. In particular, the present invention provides
compositions comprising affinity-tagged antisense rRNA molecules
corresponding to substantially all of the sequence of at least one
rRNA molecule (e.g., a eukaryotic 28S, 26S, 25S, 18S, 5.8S, and/or
5S rRNA and/or a prokaryotic 23S, 16S, and/or 5S rRNA) and methods
of using such compositions to generate rRNA-depleted samples.
[0109] Ribosomal RNA constitutes a majority mass of the RNA content
of a cell and thus, presents a tremendous background contamination
burden when studying the less abundant and, often, more relevant
transcripts. Methods to reduce rRNA are available but are severely
limiting since, for example, they require only "high quality intact
RNA" samples for good rRNA removal. Fragments of rRNA in the sample
without the complementary consensus sequences will not be removed
using the methods in the art and, thus, still contribute to
significant rRNA contamination of the sample, which limits the
efficacy of downstream transcript analysis and significantly
increases cost burden from unnecessary use of consumables and
manpower. Furthermore, even in the best case wherein the RNA in the
sample comprises so-called "intact total RNA," the sample can still
comprise a population of fragmented rRNA, which still gives rise to
a rRNA background using the methods in the art. Still further, the
methods in the art do not remove sufficient quantities of all of
the sequence exhibited by the rRNA molecules, so the rRNA
background is high when the sample is used for modern analysis
methods, such as for generating tagged sequencing templates from
RNA for use in next-generation sequencing (e.g., for so-called
"digital" gene expression methods). For example, it has been
reported that more than half of the sequence reads can be for rRNA
sequences, even after one or more rounds of rRNA removal using
commercial methods known in the art (e.g., using RiboMinus.TM.,
Life Technologies, Carlsbad, Calif.). Another requirement of these
existing methods is the need for a relatively large sample size,
which is frequently prohibitive. Often, total RNA extracted from
especially long-term stored cell/tissue samples is invariably very
fragmented and limited in quantity. Thus, a method to
simultaneously remove both fragmented and intact rRNA and to do so
from a small sample size is needed.
[0110] The present invention addresses this need as it provides,
for example, methods for the efficient removal of either completely
fragmented or variably fragmented rRNA molecules, leaving the
non-rRNA RNA transcripts generally unperturbed. Furthermore, the
method operates independently of any "unique feature" of the
non-ribosomal RNA transcripts such as, a poly(A) tail as in the
case of eukaryotic mRNA and thus, presents no selection bias in the
recovery of all non-ribosomal RNA transcripts. In certain
embodiments to achieve these objectives, one or more
affinity-tagged antisense rRNA representing substantially all of
the full-length sequence of each rRNA in whole or in part (e.g.,
eukaryotic 28S, 26S, 25S, 18S, 5.8S and/or 5Ss and/or prokaryotic
23S, 16S and 5S) are synthesized and hybridized to their respective
complementary rRNA sequences in the test sample, and the hybrids as
well as any residual unhybridized affinity-tagged antisense rRNA
molecules are then physically removed using affinity-tag-binding
molecules (e.g., linked to a binding matrix), leaving the non-rRNA
RNA transcripts unperturbed. Since substantially the entire (or the
entire) sequence of at least one, and preferably each rRNA, is
represented in the composition comprising affinity-tagged antisense
rRNA molecules, any size fragments of the native rRNA contained in
the samples are also able to form hybrids under the same
hybridization conditions and are efficiently removed along with any
intact rRNA sequences that may also be present.
[0111] In particular embodiments, in order to achieve efficient
rRNA removal, a molar excess of the affinity-tagged antisense rRNA
molecules is used in the hybridization reaction, which drives the
hybrid formation process to completion. Excess affinity-tagged
antisense rRNA molecules may also be removed since these molecules
themselves may contribute to various types of background in
different downstream methods or analyses. To accomplish this, the
affinity-tagged antisense rRNA molecules preferably contain an
optimal amount of affinity-tag molecules, and an appropriate amount
of the binding matrix comprising the affinity-tag-binding molecules
is used for removal of both rRNA-hybridized and unhybridized
affinity-tagged antisense rRNA molecules. As described in the
Examples, conditions were developed that allow the methods to be
performed using samples comprising a broader dynamic range of total
RNA than existing methods, including using samples comprising
sub-microgram quantities of total RNA.
[0112] Exemplary rRNA Sequences and Primers
[0113] The present invention includes compositions comprising
affinity-tagged antisense rRNA molecules corresponding to
substantially all of at least one rRNA molecule. The present
invention is not limited to compositions comprising antisense rRNA
molecules that exhibit sequences that are exactly complementary to
particular rRNA molecules in the sample, but instead includes
compositions comprising antisense rRNA molecules that hybridize
with or anneal to or complex with any rRNA molecules in the sample,
whether or not said rRNA molecules are from the same organism.
Exemplary Embodiment
[0114] Described below is an exemplary embodiment that may be used
to generate rRNA-depleted samples using affinity-tag-binding
molecule linked microspheres (herein after "binding microspheres"
or "microspheres"). This embodiment is not meant to limit the
present invention and instead is simply an exemplary
embodiment.
[0115] Wash the binding microspheres and suspend in solution (20 mM
Tris-HCl pH 7.5, 1 M NaCl, 1 mM EDTA and 0.0005% RNase-Free
Triton-X100). Allow the binding microspheres to reach room
temperature. Vigorously vortex the microspheres for 20 seconds to
produce a homogeneous suspension. For each reaction, pipette 25 ul
of the resuspended microspheres into a microsphere wash tube.
Centrifuge the dispensed microspheres at 10,000 rpm in a bench top
microcentrifuge for 3 minutes. Carefully pipette off and discard
the supernatant, without disturbing the microsphere pellet. Wash
the microspheres by adding 1 volume of wash solution (20 mM
Tris-HCl pH 7.5, 1 M NaCl and 1 mM EDTA) equal to the original
volume of microspheres (e.g., add 25 .mu.l of wash solution for
every 25 .mu.l of microspheres) to the tube. Resuspend the
microspheres by vigorous vortex mixing. Centrifuge the tube at
10,000 rpm for 3 minutes in a bench top microcentrifuge. Carefully
pipette off and discard the supernatant, without disturbing the
microsphere pellet. Repeat the wash, resuspension, centrifuge, and
pipetting steps.
[0116] Resuspend the microspheres in 1 volume resuspension
solution. For example, add 25 .mu.l of resuspension solution for
every 25 .mu.l of microspheres that were originally used. Resuspend
the microspheres by vigorous vortex mixing to produce a homogeneous
suspension. Add 0.25 .mu.l of ScriptGuard.TM. RNase Inhibitor
(EPICENTRE, Madison, Wis.) to the tube for every 25 .mu.l of
resuspended microspheres and store them at room temperature.
[0117] For a sample containing, for example, 50 ng-1 ug to total
RNA (e.g., human, mouse, or rat), in a 0.2-ml thin-walled
microcentrifuge tube, combine: 10.times. reaction buffer (0.5M
Tris-HCl pH7.5 and 1 M NaCl), a total RNA sample, a rRNA removal
solution (1.2 pmoles each of affinity-tagged antisense rRNA
molecules corresponding to 28S, 18S, 5.8S and 5S human rRNA
molecules), and RNase-free water. Gently mix the reaction(s) and
incubate at 68.degree. C. for 10 minutes. Remove the reaction
tube(s) to room temperature and incubate for at least 15
minutes.
[0118] Resuspend the washed microspheres from above by pipetting up
and down. For each sample, pipette 25 .mu.l of the microspheres to
a new RNase-free 1.5-ml microcentrifuge tube. Add the room
temperature total RNA sample to the appropriate tube containing the
microspheres. Mix each thoroughly by pipetting the tube contents up
and down. Incubate each tube at room temperature for 15 minutes
with occasional (every 3-4 minutes) mixing by gentle vortex (low
setting) for 5 seconds. Place the tube at 37.degree. C. for 5
minutes. Immediately centrifuge the tube at 14,000 rpm in a
microcentrifuge for 5 minutes at room temperature. Carefully
pipette off each supernatant, which contains the rRNA-depleted
sample, to an RNase-free 1.5-ml microcentrifuge tube. If there are
microspheres still visible in the sample, and ethanol precipitation
is desired to be used, repeat the above procedure. The
rRNA-depleted sample can, for example, be purified by ethanol
precipitation or column method.
[0119] For ethanol precipitation, adjust the volume of each sample
to 180 .mu.l using RNase-free water. Add 18 .mu.l of 3M Sodium
Acetate to each tube. Add 2 .mu.l of Glycogen (10 mg/ml) to each
tube and mix by gentle vortex. Add 3 volumes (600 .mu.l) of ice
cold 100% ethanol to each tube and mix thoroughly by gentle vortex.
Place the tubes at -20.degree. C. for at least 1 hour. Centrifuge
the tubes at >10,000.times.g in a microcentrifuge for 30
minutes. Carefully remove and discard the supernatant. Wash the
pellet with ice cold 70% ethanol and centrifuge at
>10,000.times.g for 5 minutes. Carefully remove and discard the
supernatant. Repeat the ethanol and centrifuge step. Centrifuge
briefly to collect any residual supernatant. Carefully remove and
discard the supernatant and allow the pellet to air dry at room
temperature for 5 minutes. Dissolve the pellet in the desired
volume of RNase-free water or buffer.
[0120] For column purification, the RNA Clean &
Concentrator.TM.5 Column (Zymo Research; Catalog Numbers R1015,
R1016) can be used to used to purify the rRNA-depleted RNA sample.
If using the RNA Clean & Concentrator-5 Column, follow the
manufacturer's procedure entitled "To recover total RNA including
small RNAs". The eluted RNA can be used immediately of stored at
-70.degree. C. to -80.degree. C.
[0121] Sequencing Technologies
[0122] Purified RNA samples generated in accordance with the
present invention can be sequenced using any type of suitable
sequencing technology. The present invention is not limited by the
type of sequencing method employed. Exemplary sequencing methods
are described below.
[0123] Illustrative non-limiting examples of nucleic acid
sequencing techniques include, but are not limited to, chain
terminator (Sanger) sequencing and dye terminator sequencing. Chain
terminator sequencing uses sequence-specific termination of a DNA
synthesis reaction using modified nucleotide substrates. Extension
is initiated at a specific site on the template DNA by using a
short radioactive, or other labeled, oligonucleotide primer
complementary to the template at that region. The oligonucleotide
primer is extended using a DNA polymerase, standard four
deoxynucleotide bases, and a low concentration of one chain
terminating nucleotide, most commonly a di-deoxynucleotide. This
reaction is repeated in four separate tubes with each of the bases
taking turns as the di-deoxynucleotide. Limited incorporation of
the chain terminating nucleotide by the DNA polymerase results in a
series of related DNA fragments that are terminated only at
positions where that particular di-deoxynucleotide is used. For
each reaction tube, the fragments are size-separated by
electrophoresis in a slab polyacrylamide gel or a capillary tube
filled with a viscous polymer. The sequence is determined by
reading which lane produces a visualized mark from the labeled
primer as you scan from the top of the gel to the bottom.
[0124] Dye terminator sequencing alternatively labels the
terminators. Complete sequencing can be performed in a single
reaction by labeling each of the di-deoxynucleotide
chain-terminators with a separate fluorescent dye, which fluoresces
at a different wavelength.
[0125] A set of methods referred to as "next-generation sequencing"
techniques have emerged as alternatives to Sanger and
dye-terminator sequencing methods (Voelkerding et al., Clinical
Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol.,
7: 287-296; each herein incorporated by reference in their
entirety). Most current methods describe the use of next-generation
sequencing technology for de novo sequencing of whole genomes to
determine the primary nucleic acid sequence of an organism. In
addition, targeted re-sequencing (deep sequencing) allows for
sensitive mutation detection within a population of wild-type
sequence. Some examples include recent work describing the
identification of HIV drug-resistant variants as well as EGFR
mutations for determining response to anti-TK therapeutic drugs.
Recent publications describing the use of bar code primer sequences
permit the simultaneous sequencing of multiple samples during a
typical sequencing run including, for example: Margulies, M. et al.
"Genome Sequencing in Microfabricated High-Density Picolitre
Reactors", Nature, 437, 376-80 (2005); Mikkelsen, T. et al.
"Genome-Wide Maps of Chromatin State in Pluripotent and
Lineage-Committed Cells", Nature, 448, 553-60 (2007); McLaughlin,
S. et al. "Whole-Genome Resequencing with Short Reads: Accurate
Mutation Discovery with Mate Pairs and Quality Values", ASHG Annual
Meeting (2007); Shendure J. et al. "Accurate Multiplex Polony
Sequencing of an Evolved Bacterial Genome", Science, 309, 1728-32
(2005); Harris, T. et al. "Single-Molecule DNA Sequencing of a
Viral Genome", Science, 320, 106-9 (2008); Simen, B. et al.
"Prevalence of Low Abundance Drug Resistant Variants by Ultra Deep
Sequencing in Chronically HIV-infected Antiretroviral (ARV) Naive
Patients and the Impact on Virologic Outcomes", 16th International
HIV Drug Resistance Workshop, Barbados (2007); Thomas, R. et al.
"Sensitive Mutation Detection in Heterogeneous Cancer Specimens by
Massively Parallel Picoliter Reactor Sequencing", Nature Med., 12,
852-855 (2006); Mitsuya, Y. et al. "Minority Human Immunodeficiency
Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse
Transcriptase Codon 215 Revertant Mutations", J. Vir., 82,
10747-10755 (2008); Binladen, J. et al. "The Use of Coded PCR
Primers Enables High-Throughput Sequencing of Multiple Homolog
Amplification Products by 454 Parallel Sequencing", PLoS ONE, 2,
e197 (2007); and Hoffmann, C. et al. "DNA Bar Coding and
Pyrosequencing to Identify Rare HIV Drug Resistance Mutations",
Nuc. Acids Res., 35, e91 (2007), all of which are herein
incorporated by reference.
[0126] Compared to traditional Sanger sequencing, next-gen
sequencing technology produces large amounts of sequencing data
points. A typical run can easily generate tens to hundreds of
megabases per run, with a potential daily output reaching into the
gigabase range. This translates to several orders of magnitude
greater than a standard 96-well plate, which can generate several
hundred data points in a typical multiplex run. Target amplicons
that differ by as little as one nucleotide can easily be
distinguished, even when multiple targets from related species or
organisms are present. This greatly enhances the ability to do
accurate genotyping. Next-gen sequence alignment software programs
used to produce consensus sequences can easily identify novel point
mutations, which could result in new strains with associated drug
resistance. The use of primer bar coding also allows multiplexing
of different patient samples within a single sequencing run.
[0127] Next-generation sequencing (NGS) methods share the common
feature of massively parallel, high-throughput strategies, with the
goal of lower costs in comparison to older sequencing methods. NGS
methods can be broadly divided into those that require template
amplification and those that do not. Amplification-requiring
methods include pyrosequencing commercialized by Roche as the 454
technology platforms (e.g., GS 20 and GS FLX), the Solexa platform
commercialized by Illumina, and the Supported Oligonucleotide
Ligation and Detection (SOLiD) platform commercialized by Applied
Biosystems. Non-amplification approaches, also known as
single-molecule sequencing, are exemplified by the HeliScope
platform commercialized by Helicos BioSciences, and emerging
platforms commercialized by VisiGen and Pacific Biosciences,
respectively.
[0128] In pyrosequencing (Voelkerding et al., Clinical Chem., 55:
641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296;
U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; each herein
incorporated by reference in its entirety), template DNA is
fragmented, end-repaired, ligated to adaptors, and clonally
amplified in-situ by capturing single template molecules with beads
bearing oligonucleotides complementary to the adaptors. Each bead
bearing a single template type is compartmentalized into a
water-in-oil microvesicle, and the template is clonally amplified
using a technique referred to as emulsion PCR. The emulsion is
disrupted after amplification and beads are deposited into
individual wells of a picotitre plate functioning as a flow cell
during the sequencing reactions. Ordered, iterative introduction of
each of the four dNTP reagents occurs in the flow cell in the
presence of sequencing enzymes and luminescent reporter such as
luciferase. In the event that an appropriate dNTP is added to the
3' end of the sequencing primer, the resulting production of ATP
causes a burst of luminescence within the well, which is recorded
using a CCD camera. It is possible to achieve read lengths greater
than or equal to 400 bases, and 1.times.10.sup.6 sequence reads can
be achieved, resulting in up to 500 million base pairs (Mb) of
sequence.
[0129] In the Solexa/Illumina platform (Voelkerding et al.,
Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.
Microbiol., 7: 287-296; U.S. Pat. No. 6,833,246; U.S. Pat. No.
7,115,400; U.S. Pat. No. 6,969,488; each herein incorporated by
reference in its entirety), sequencing data are produced in the
form of shorter-length reads. In this method, single-stranded
fragmented DNA is end-repaired to generate 5'-phosphorylated blunt
ends, followed by Klenow-mediated addition of a single A base to
the 3' end of the fragments. A--addition facilitates addition of
T--overhang adaptor oligonucleotides, which are subsequently used
to capture the template-adaptor molecules on the surface of a flow
cell that is studded with oligonucleotide anchors. The anchor is
used as a PCR primer, but because of the length of the template and
its proximity to other nearby anchor oligonucleotides, extension by
PCR results in the "arching over" of the molecule to hybridize with
an adjacent anchor oligonucleotide to form a bridge structure on
the surface of the flow cell. These loops of DNA are denatured and
cleaved. Forward strands are then sequenced with reversible dye
terminators. The sequence of incorporated nucleotides is determined
by detection of post-incorporation fluorescence, with each fluor
and block removed prior to the next cycle of dNTP addition.
Sequence read length ranges from 36 nucleotides to over 50
nucleotides, with overall output exceeding 1 billion nucleotide
pairs per analytical run.
[0130] Sequencing nucleic acid molecules using SOLiD technology
(Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et
al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148;
U.S. Pat. No. 6,130,073; each herein incorporated by reference in
their entirety) also involves fragmentation of the template,
ligation to oligonucleotide adaptors, attachment to beads, and
clonal amplification by emulsion PCR. Following this, beads bearing
template are immobilized on a derivatized surface of a glass
flow-cell, and a primer complementary to the adaptor
oligonucleotide is annealed. However, rather than utilizing this
primer for 3' extension, it is instead used to provide a 5'
phosphate group for ligation to interrogation probes containing two
probe-specific bases followed by 6 degenerate bases and one of four
fluorescent labels. In the SOLiD system, interrogation probes have
16 possible combinations of the two bases at the 3' end of each
probe, and one of four fluors at the 5' end. Fluor color and thus
identity of each probe corresponds to specified color-space coding
schemes. Multiple rounds (usually 7) of probe annealing, ligation,
and fluor detection are followed by denaturation, and then a second
round of sequencing using a primer that is offset by one base
relative to the initial primer. In this manner, the template
sequence can be computationally re-constructed, and template bases
are interrogated twice, resulting in increased accuracy. Sequence
read length averages 35 nucleotides, and overall output exceeds 4
billion bases per sequencing run.
[0131] In certain embodiments, nanopore sequencing in employed
(see, e.g., Astier et al., J Am Chem Soc. 2006 Feb. 8;
128(5):1705-10, herein incorporated by reference). The theory
behind nanopore sequencing has to do with what occurs when the
nanopore is immersed in a conducting fluid and a potential
(voltage) is applied across it: under these conditions a slight
electric current due to conduction of ions through the nanopore can
be observed, and the amount of current is exceedingly sensitive to
the size of the nanopore. If DNA molecules pass (or part of the DNA
molecule passes) through the nanopore, this can create a change in
the magnitude of the current through the nanopore, thereby allowing
the sequences of the DNA molecule to be determined.
[0132] HeliScope by Helicos BioSciences (Voelkerding et al.,
Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.
Microbiol., 7: 287-296; U.S. Pat. No. 7,169,560; U.S. Pat. No.
7,282,337; U.S. Pat. No. 7,482,120; U.S. Pat. No. 7,501,245; U.S.
Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S. Pat. No.
7,501,245; each herein incorporated by reference in their entirety)
is the first commercialized single-molecule sequencing platform.
This method does not require clonal amplification. Template DNA is
fragmented and polyadenylated at the 3' end, with the final
adenosine bearing a fluorescent label. Denatured polyadenylated
template fragments are ligated to poly(dT) oligonucleotides on the
surface of a flow cell. Initial physical locations of captured
template molecules are recorded by a CCD camera, and then label is
cleaved and washed away. Sequencing is achieved by addition of
polymerase and serial addition of fluorescently-labeled dNTP
reagents. Incorporation events result in fluor signal corresponding
to the dNTP, and signal is captured by a CCD camera before each
round of dNTP addition. Sequence read length ranges from 25-50
nucleotides, with overall output exceeding 1 billion nucleotide
pairs per analytical run. Other emerging single molecule sequencing
methods real-time sequencing by synthesis using a VisiGen platform
(Voelkerding et al., Clinical Chem., 55: 641-658, 2009; U.S. Pat.
No. 7,329,492; U.S. patent application Ser. No. 11/671,956; U.S.
patent application Ser. No. 11/781,166; each herein incorporated by
reference in their entirety) in which immobilized, primed DNA
template is subjected to strand extension using a
fluorescently-modified polymerase and florescent acceptor
molecules, resulting in detectible fluorescence resonance energy
transfer (FRET) upon nucleotide addition. Another real-time single
molecule sequencing system developed by Pacific Biosciences
(Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et
al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 7,170,050;
U.S. Pat. No. 7,302,146; U.S. Pat. No. 7,313,308; U.S. Pat. No.
7,476,503; all of which are herein incorporated by reference)
utilizes reaction wells 50-100 nm in diameter and encompassing a
reaction volume of approximately 20 zeptoliters
(10.times.10.sup.-21 L). Sequencing reactions are performed using
immobilized template, modified phi29 DNA polymerase, and high local
concentrations of fluorescently labeled dNTPs. High local
concentrations and continuous reaction conditions allow
incorporation events to be captured in real time by fluor signal
detection using laser excitation, an optical waveguide, and a CCD
camera.
[0133] Binding Matrix Solid Supports
[0134] The binding matrix employed in the present invention may be
any surface or solid support to which the affinity-tag-binding
molecule can be attached and which does not exhibit substantial
non-specific binding of the affinity-tagged antisense rRNA
molecules. The present invention is not limited to any one solid
support. In some embodiments, polystyrene plates containing either
96 or 384 wells are employed. In some embodiments, streptavidin
(SA) coated 96-well or 384-well microtiter plates (Boehringer
Mannheim Biochemicals, Indianapolis, Ind.) are used as solid
supports. In some embodiments, particles or beads are employed. The
particles can be made of any suitable material, including, but not
limited to, latex. In some embodiments, minicolumns are employed.
The columns may contain affinity-tag-binding molecules. In other
embodiments, a BEADARRAY (Illumina, San Diego, Calif.) is employed.
A variety of other solid supports are contemplated including, but
not limited to, glass microscope slides, glass wafers, gold,
silicon, microchips, and other plastic, metal, ceramic, or
biological surfaces.
EXAMPLES
[0135] It is understood that the examples and embodiments described
herein are for illustrative purposes only and are not intended to
limit the scope of the claimed invention. It is also understood
that various modifications or changes in light the examples and
embodiments described herein will be suggested to persons skilled
in the art and are to be included within the spirit and purview of
this application and scope of the appended claims.
Example 1
Synthesis of Biotinylated Antisense rRNA Molecules Representing the
Full-Length Sequences of Ribosomal RNA (rRNA) Molecules of
Eukaryotes and Prokaryotes
[0136] In order to synthesize antisense rRNA corresponding to the
complete sequence of each rRNA molecule, PCR templates were made
containing a phage promoter sequence in all but one case as
follows.
Human 18S, 5.8S and 5S rRNA PCR Templates for In Vitro Synthesis of
Antisense rRNA
[0137] PCR amplicons representing the full-length 18S (Accession#
NR003286), 5.8S (Accession# U13369 REGION: 6623-6779) and 5S
(Accession # NR 023363) rRNA sequences were synthesized. Each
100-.mu.l PCR reaction comprised 20 pmoles of a forward primer, 20
pmoles of a reverse primer containing T7 RNA polymerase phage
promoter sequence (shown in italics) and 5 ng random primed
first-strand cDNA made from Universal Human Reference total RNA
(Stratagene) under FailSafe PCR reaction conditions optimized as
described by the manufacturer (EPICENTRE Biotechnologies). The PCR
amplification reactions were performed for 25 cycles to 30 cycles
depending on the primer pair and the PCR amplicons corresponding to
the full-length 18S, 5.8S and 5S rRNA sequences and PCR products
were purified using the Qiaquick PCR purification kit as
recommended by the manufacturer (Qiagen). The following is a list
of the respective forward and reverse primers used:
TABLE-US-00001 18S rRNA forward primer (SEQ ID NO: 1:
5'-CCTACCTACCTGGTTGATCC) and reverse primer (SEQ ID NO: 2:
5'-AATTCTAATACGACTCACTATAGGGAGAGATC CTTCCGCAGGTTCACCTAC). 5.8S rRNA
forward primer (SEQ ID NO: 3: 5'-CGACTCTTAGCGGTGGATCACTC) and
reverse primer (SEQ ID NO: 4: 5'-AATTCTAATACGACTCACTATAGGGAGAGATC
CTTCCGCAGGTTCACCTAC) 5S rRNA forward primer (SEQ ID NO: 5:
5'-GTCTACGGCCATACCACCCTGAA) and reverse primer (SEQ ID NO: 6:
5'-AATTCTAATACGACTCACTATAGGGAGAAAG CCTACAGCACCCGGTATTC)
Escherichia coli 23S, 16S and 5S rRNA PCR Templates for In Vitro
Synthesis of Antisense rRNA
[0138] PCR amplicons representing the full-length 23S (Accession#
EG30077), 16S (Accession# EG30084) and 5S (Accession # EG30070)
rRNA sequences were synthesized. Each 100 .mu.l PCR reaction
comprised 20 pmoles of a forward primer, 20 pmoles of a reverse
primer containing T7 RNA polymerase phage promoter sequence (shown
in italics) and 5 ng random primed first-strand cDNA made from E.
coli K-12 total RNA under FailSafe PCR reaction conditions
optimized as described by the manufacturer (EPICENTRE
Biotechnologies). The PCR amplification reactions were performed
for 25 cycles to 30 cycles depending on the primer pair and the PCR
amplicons corresponding to the full-length 23S, 16S and 5S rRNA
sequences and PCR products were purified using the Qiaquick PCR
purification kit as recommended by the manufacturer (Qiagen). The
following is a list of the respective forward and reverse primers
used:
TABLE-US-00002 23S rRNA In order to efficiently amplify the
complete 23S rRNA sequence, two pairs of primers were used whereby,
the full-length 23S rRNA sequence was divided into approximately
two equal segments. The 5' 23S rRNA segment comprised forward
primer (SEQ ID NO: 7: 5'-AAGCGACTAAGCGTACACGGTGGA) and reverse
primer (SEQ ID NO: 8: 5'-AATTCTAATACGACTCACTATAGGGAGATTCC
TGGAAGCAGGGCATTTGTTG) and the 3' 23S rRNA segment comprised forward
primer (SEQ ID NO: 9: 5'-CAACAAATGCCCTGCTTCCAGGAA) and reverse
primer (SEQ ID NO: 10: 5'-AATTCTAATACGACTCACTATAGGGAGACAC
GGTTCATTAGTACCGGTTAGCT). 16S rRNA forward primer (SEQ ID NO: 11:
5'-AGAGTTTGATCCTGGCTCAG) and reverse primer (SEQ ID NO: 12:
5'-AATTCTAATACGACTCACTATAGGGAGAGGA GGTGATCCAACCGCAGGTT). 5S rRNA
forward primer (SEQ ID NO: 13: 5'-TGCCTGGCGGCAGTAGCGCGGT) and
reverse primer (SEQ ID NO: 14: 5'-AATTCTAATACGACTCACTATAGGGAGATGC
CTGGCAGTTCCCTACTCTC).
Each PCR amplicon (300 ng) representing the respective human or E.
coli rRNA sequence was analyzed by ethidium bromide stained agarose
gel electrophoresis as shown in FIG. 1A.
Human 28S Ribosomal RNA Clone
[0139] PCR amplification of the complete 28S rRNA (Accession#
NR003287) sequence was found not to be very efficient, especially
for the approximately 1200-nt 5'-end sequence, even after dividing
the full-length sequence into segments ranging from 1 Kb to 2 Kb.
Thus, in order to generate the required template for in vitro
synthesis of the 28S antisense rRNA, the 28S rRNA sequence was
cloned into both a T7 phage promoter-containing plasmid (pSP73;
Promega Corporation) and a SP6 phage promoter-containing plasmid
(pSP64; Promega Corporation) using standard cloning methods and
strategies (Maniatis et. al. (1982) Molecular Cloning, A Laboratory
Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and
information described by Maden et. al. (1987) Biochem. J. 246:
519-527.
In Vitro Synthesis of Biotinylated Antisense rRNA Molecules from
the Respective PCR Templates for Human 18S, 5.8S and 5S and E. coli
23S, 16S and 5S rRNA Sequences
[0140] Either an AmpliScribe.TM. T7 High Yield Transcription Kit or
an AmpliScribe.TM. T7-FLASH Transcription Kit (EPICENTRE, Madison,
Wis.) was used for in vitro transcription (IVT) reactions
comprising 500 ng to 1000 ng of the respective templates (human
18S, 5.8S and 5S and E. coli 23S, 16S and 5S) were performed as
described by the manufacturer (EPICENTRE Biotechnologies) with the
following modification--the uridine triphosphate (UTP) was replaced
with mixtures of biotin-16-UTP and UTP comprising 10%, 20%, 35%,
50% or 60% biotin-16-UTP. The IVT reactions were incubated at
37.degree. C. for 4 to 6 hours and the DNA template used in each
reaction was then digested with DNase I as recommended by the
manufacturer. Each biotinylated antisense rRNA was subsequently
purified using illustra RNAspin Mini columns as recommended by the
manufacturer (GE Healthcare) and eluted in RNase-free water. Each
purified antisense rRNA was treated with Baseline-Zero.TM. DNase I
as recommended by the manufacturer (EPICENTRE Biotechnologies) to
remove all traces of the DNA template used for transcription. The
biotinylated antisense rRNA was again purified using illustra
RNAspin Mini columns, eluted in RNase-free water and the RNA
concentration determined by measuring the absorbance at 260 nm with
a spectrophotometer. (Note: subsequent experiments showed that best
results for generating rRNA-depleted samples with respect to 5.8S
and 5S eukaryotic rRNA or 5S prokaryotic rRNA were obtained using
at least about 75% biotin-16-UTP in the in vitro transcription
reaction.)
[0141] Each purified antisense RNA (300 ng), representing the
entire human or E. coli rRNA sequence, as analyzed by ethidium
bromide stained agarose gel electrophoresis, is shown in FIG.
1B.
In Vitro Synthesis of Biotinylated Antisense rRNA from the Plasmid
Templates for Human 28S rRNA Sequence
[0142] Initially, 1 .mu.g of the linearized T7-28S rRNA plasmid
clone was used in an AmpliScribe.TM. T7 High Yield Transcription
Kit for in vitro transcription (IVT) as described by the
manufacturer (EPICENTRE Biotechnologies) with the following
modification--a 1:1 mixture of bio-UTP:UTP instead of 100% UTP.
Incubation was performed at 37.degree. C. for 4 hours and the DNA
template then removed by digesting with DNase I as recommended by
the manufacturer (EPICENTRE Biotechnologies). An aliquot (300 ng)
of the purified transcription reaction product was analyzed by gel
electrophoresis. It was evident from the results (FIG. 1C, Lane 1)
that very little, if any of the expected full-length antisense 28S
rRNA was produced in the IVT reaction. Instead, RNA molecules
ranging in size from low to high molecular weights were observed.
Thus, it appeared that the T7 RNA polymerase was unable to
efficiently transcribe the 28S DNA template.
[0143] Next, 1 .mu.g of the linearized SP6-28S rRNA plasmid clone
was tested in a standard AmpliScribe.TM. SP6 High Yield
Transcription Kit for in vitro transcription (IVT) as described by
the manufacturer (EPICENTRE Biotechnologies) using a similar 1:1
mixture of bio-UTP and UTP. Incubation was performed at 37.degree.
C. for 4 hours and the DNA template then removed by digesting with
DNase I as recommended by the manufacturer (EPICENTRE
Biotechnologies). The biotinylated antisense rRNA produced was
subsequently purified using illustra RNAspin Mini columns as
recommended by the manufacturer (GE Healthcare) and eluted in
RNase-free water. Thereafter, the purified antisense 28S rRNA was
treated with Baseline-Zero.TM. DNase I as recommended by the
manufacturer (EPICENTRE Biotechnologies) to remove all traces of
the DNA template used for transcription. The biotinylated antisense
rRNA was again purified using illustra RNAspin Mini columns, eluted
in RNase-free water and the RNA concentration determined by
measuring the absorbance at 260 nm with a spectrophotometer. An
aliquot (300 ng) of the transcription reaction was analyzed by gel
electrophoresis and it was evident from the results that the
expected antisense 28S rRNA was produced in the SP6-IVT reaction
(FIG. 1C, Lane 2).
Example 2
Removal of Biotinylated Antisense rRNA and Resulting ds-rRNA
Hybrids Formed Using E. coli Total RNA Using Streptavidin-Coated
Microspheres
[0144] E. coli rRNA was used as the model to test the amount of
ProActive.RTM. Streptavidin Coated Microspheres (Bangs
Laboratories) needed to remove a fixed quantity biotinylated
antisense rRNA from solution. The streptavidin-coated microspheres
were washed and resuspended in the bind/wash buffer as recommended
by the manufacturer (Bangs Laboratories). E. coli biotinylated
antisense rRNA materials synthesized using 35% biotin-UTP as
described in Example 1 were used to prepare a mixture comprising
one microgram each of the 23 S rRNA 5' segment, the 23S rRNA 3'
segment and the full-length 16S rRNA in a 4-.mu.l volume of
RNase-free water. Each 4-.mu.l biotinylated antisense rRNA mixture
would represent at least a >2-fold molar excess when mixed with
one microgram of native E. coli total RNA, which concentration was
selected to drive the hybridization of the sense and antisense rRNA
to completion or near completion when mixed together.
[0145] The amount of streptavidin-coated microspheres required to
efficiently remove the added biotinylated antisense rRNA added to a
hybridization reaction was determined. Three 4-.mu.l aliquots of
the E. coli biotinylated antisense rRNA mixture were prepared in
0.2 ml microcentrifuge tubes to a final reaction volume of 20 .mu.l
comprising 1.times. hybridization buffer (50 mM Tris-HCl, pH7.5 and
100 mM NaCl). The reactions were incubated at 68.degree. C. for 5
minutes and then at room temperature for 15 minutes. Reactions #1
and #2 were each then added to a fresh 1.5-ml microcentrifuge tube
containing 20 .mu.l of the washed and resuspended
streptavidin-coated microspheres. Control reaction #3 was added to
a fresh 1.5-ml microcentrifuge tube with only 20 .mu.l bind/wash
buffer. All reactions were further incubated at room temperature
for 15 minutes with occasional gentle mixing (3-4 minutes) in order
to allow formation of the biotin-streptavidin complex. The
biotinylated antisense rRNA remaining in solution following capture
by the microspheres was then purified using RNA Clean-up Kit.TM.-5
columns as recommended by the manufacturer (ZYMO Research).
Reactions #1 and #3 were added directly in the ZYMO RNA Clean-up
procedure whereas, reaction #2 was first spun at 12,000 rpm for 5
minutes using a benchtop centrifuge to pellet the microspheres and
the supernatant removed and then added to the ZYMO RNA Clean-up
procedure. Each RNA sample was eluted in 10 .mu.l RNase-free water
and a 5-.mu.l aliquot analyzed by ethidium bromide stained agarose
gel electrophoresis as shown in FIG. 2A.
[0146] FIG. 2A, lanes 1 and 2 show the residual biotinylated
antisense rRNA following binding to the streptavidin coated
microspheres compared to the control (Lane 3). The amount of
antisense RNA remaining was more pronounced in Lane 2 where the
microspheres were removed prior to the ZYMO RNA clean-up procedure.
While unclear, it is possible that adding the hybridization
reaction mixture with the microspheres directly to the RNA
purification column reduced the binding capacity of the column
resulting in a further loss of the biotinylated antisense RNA.
Nevertheless, it was evident from this experiment that 20 .mu.l of
the streptavidin coated microspheres was insufficient to remove the
entire amount of biotinylated antisense rRNA used.
[0147] Thus, an additional 20 .mu.l of washed microspheres was
tested to determine if it would be sufficient to remove the 4 .mu.l
of biotinylated antisense rRNA. Four reactions, each comprising 4
.mu.l of the E. coli biotinylated antisense rRNA were prepared in
1.times. hybridization buffer to a final volume of 20 .mu.l. The
reactions were incubated at 68.degree. C. for 5 minutes and then at
room temperature for 15 minutes. Each of reactions #2 and #3 was
added to a fresh 1.5-ml microcentrifuge tube containing 20 .mu.l of
the washed and resuspended streptavidin-coated microspheres.
Reaction #4 was added to a fresh 1.5-ml microcentrifuge tube
containing 40 .mu.l of the washed and resuspended
streptavidin-coated microspheres. Control reaction #1 was added to
a fresh 1.5-ml microcentrifuge tube with only bind/wash buffer. The
reactions were further incubated at room temperature for 15 minutes
with occasional gentle mixing (3-4 minutes) in order to allow
formation of the biotin-streptavidin complex. The reactions were
spun at 12,000 rpm for 5 minutes using a benchtop centrifuge to
pellet the microspheres and each supernatant transferred to a fresh
1.5-ml microcentrifuge tubes. To the supernatant of reaction #3, an
additional 20 .mu.l of the washed streptavidin-coated microspheres
was added and again incubated at room temperature with occasional
gentle mixing (3-4 minutes). Reaction #3 was centrifuged at 12,000
rpm for 5 minutes once more and the supernatant transferred to a
fresh 1.5-ml microcentrifuge tube. The biotinylated antisense RNA
contained in each of the four reactions was purified using the ZYMO
RNA Clean-up procedure and eluted in 10 .mu.l RNase-free water. The
entire eluate for each was analyzed by ethidium bromide stained
agarose gel electrophoresis as shown in FIG. 2B.
[0148] FIG. 2B, lane 2 again shows that biotinylated antisense rRNA
is not completely removed when only 20 .mu.l of microspheres is
used. However, when either 20 .mu.l of microspheres followed by a
second 20 .mu.l of microspheres or 40 .mu.l of microspheres was
used, there appears to be complete removal of the added
biotinylated antisense rRNA (Lane 3 and Lane 4, respectively).
Thus, the one treatment with 40 .mu.l of microspheres was generally
preferred since it required fewer steps.
[0149] Next, the amount of microspheres used as described in FIG.
2B was tested to determine if it was also sufficient to efficiently
remove the double-stranded ribosomal RNA (ds-rRNA) hybrids formed
after annealing the biotinylated antisense rRNA and the native rRNA
contained in total RNA. Firstly, in order to demonstrate the
formation of the ds-rRNA hybrid, 500 ng of E. coli total RNA was
mixed with 4 .mu.l of the E. coli biotinylated antisense rRNA in a
reaction comprising 1.times. hybridization buffer in a final volume
of 20 .mu.l. The reaction was incubated at 68.degree. C. for 5
minutes and then at room temperature for 15 minutes, purified using
the ZYMO RNA Clean-up procedure and eluted in 10 .mu.l RNase-free
water. A 5-.mu.l aliquot was analyzed by ethidium bromide stained
agarose gel electrophoresis along with equivalent amounts of the
individual E. coli total RNA and biotinylated antisense rRNA as
shown in FIG. 2C. FIG. 2C, lane 3 shows the efficient formation of
the ds-rRNA hybrids compared to the individual sense and antisense
RNA samples (Lanes 1 and 2, respectively).
[0150] Next, the removal of the ds-rRNA hybrids was tested using
the quantities of washed microspheres described in FIG. 2B. Three
reactions (#1, #2 and #3), each comprising 500 ng of E. coli total
RNA and 4 .mu.l of the E. coli biotinylated antisense rRNA in
1.times. hybridization buffer in a final volume of 20 .mu.l were
prepared, along with two control reactions--one containing only the
E. coli biotinylated antisense rRNA (#4) and the other, only the E.
coli total RNA (#5). The reactions were incubated at 68.degree. C.
for 5 minutes and then at room temperature for 15 minutes.
Reactions #1 and #3 were each added to a fresh 1.5-ml
microcentrifuge tube containing 20 .mu.l of the washed and
resuspended streptavidin-coated microspheres. Reactions #2 and #4
were each added to a fresh 1.5-ml microcentrifuge tube containing
40 .mu.l of the washed and resuspended streptavidin-coated
microspheres. Control reaction #5 was added to a fresh 1.5-ml
microcentrifuge tube containing only bind/wash buffer. The
reactions were further incubated at room temperature for 15 minutes
with occasional gentle mixing (3-4 minutes) in order to allow
formation of the biotin-streptavidin complex. The reactions were
spun at 12,000 rpm for 5 minutes using a benchtop centrifuge to
pellet the microspheres and each supernatant transferred to a fresh
1.5-ml microcentrifuge tubes. To the supernatant of reaction #3, an
additional 20 .mu.l of the washed streptavidin-coated microspheres
was added and incubated at room temperature with occasional gentle
mixing (3-4 minutes). Reaction #3 was again centrifuged at 12,000
rpm for 5 minutes and the supernatant transferred to a fresh 1.5-ml
microcentrifuge tube. The RNA contained in each of the five
reactions was purified using the ZYMO RNA Clean-up procedure and
eluted in 10 .mu.l RNase-free water. A 5-.mu.l aliquot of each was
analyzed by ethidium bromide stained agarose gel electrophoresis as
shown in FIG. 2D. In addition, 250 ng of untreated E. coli total
RNA was run as a control.
[0151] FIG. 2D, Lane 1 shows the residual ds-rRNA when only 20
.mu.l of microspheres was used. However, with either 2.times.20
.mu.l (Lane 2) or 1.times.40 .mu.l of microspheres (Lane 3), the
ds-rRNA hybrids were no longer visible indicating that the
biotinylated antisense rRNA was capable of annealing to its
complementary sense rRNA sequences and facilitate their removal by
the streptavidin microspheres. The quantity of E. coli total RNA
did not appear to be affected by the addition of 40 .mu.l of
microspheres (Lane 5) when compared to a similar amount of
untreated E. coli total RNA (Lane 6). Thus, it was evident that 40
.mu.l of microspheres was sufficient to remove either the added
biotinylated antisense rRNA or the resulting ds-rRNA hybrids formed
after hybridization of the biotinylated antisense rRNA to the
rRNA.
Example 3
Effect of Using Different Levels of Biotin-UTP for Synthesis of
Biotinylated Antisense rRNA and its Removal with the Streptavidin
Coated Microspheres
[0152] The experiments described in Example 2 above used
biotinylated antisense rRNA synthesized using a UTP:biotin-UTP
mixture comprising 35% biotin-UTP. For the amount of antisense rRNA
used in the hybridization reaction, 40 .mu.l of the strepavidin
microspheres was needed for its efficient removal. Thus, it was
thought that antisense rRNA made with lesser amounts of biotin-UTP
should require less streptavidin microspheres for its quantitative
removal, which would result in an overall reduction in the cost
associated with the amount of both the biotin-UTP and microspheres
required. Biotinylated antisense 16S and 23S rRNA were prepared
using either 10% or 20% biotin-UTP solutions as described above in
Example 1 and standard 4-.mu.l mixtures of the respective
biotinylated antisense rRNA were then made. The percentage of
biotin-UTP used in the in vitro transcription reaction for its
synthesis is used to refer to the biotinylated antisense rRNA
product; for example, if the percentage of biotin-UTP used in the
in vitro transcription reaction is 10%, with the remaining 90%
being UTP, the product is referred to herein as 10% biotinylated
antisense rRNA, even though the percentage of biotin-UTP
nucleotides incorporated into the product may be less than 10%
compared to the UTP nucleotides incorporated into the product.
[0153] Two sets of three reactions each comprising 4 .mu.l of
either the 10% (reaction #1, #2 and #3) or 20% (reactions #4, #5
and #6) biotinylated antisense rRNA mixture were prepared in a
final volume of 20 .mu.l in 1.times. hybridization buffer. The
reactions were incubated at 68.degree. C. for 5 minutes and then at
room temperature for 15 minutes. Reactions # 2 and #5 were each
transferred to a fresh 1.5-ml microcentrifuge tube containing 20
.mu.l of washed streptavidin microspheres and reactions # 3 and #6
were each transferred to a fresh 1.5-ml microcentrifuge tube
containing 40 .mu.l of washed streptavidin microspheres. Reactions
#1 and #4 were each transferred to a fresh 1.5-ml microcentrifuge
tubes containing 40 .mu.l of bind/wash buffer. The reactions were
further incubated at room temperature for 15 minutes with
occasional gentle mixing (3-4 minutes) in order to allow formation
of the biotin-streptavidin complex. The reactions were spun at
12,000 rpm for 5 minutes on a benchtop centrifuge and the
supernatant transferred to fresh 1.5-ml microcentrifuge tubes. Any
biotinylated antisense rRNA remaining in the supernatants were
purified using the ZYMO RNA Clean-up procedure and eluted in 10
.mu.l RNase-free water. The entire amount of each purified sample
was analyzed by ethidium bromide stained agarose gel
electrophoresis as shown in FIG. 3A.
[0154] FIG. 3A, Lanes 1 and 4 show the 10% and 20% biotinylated
antisense rRNA not treated with microspheres, respectively. Lanes 2
and 3 show the 10% biotinylated antisense rRNA treated with 20
.mu.l and 40 .mu.l of microspheres, respectively. Lanes 5 and 6
show the 20% biotinylated antisense rRNA treated with 20 .mu.l and
40 .mu.l of microspheres, respectively. Clearly, there was a
significant amount of both the 10% and 20% biotinylated antisense
rRNA remaining independent of either 20 .mu.l or 40 .mu.l of
microspheres used (Lanes 2, 3, 5 and 6). Nevertheless, the 20%
condition (Lanes 5 and 6) was clearly better than the 10% condition
(Lanes 2 and 3). Thus, it appeared that using these lower levels of
biotin-UTP were used to synthesize the biotinylated rRNA, at least
some of the antisense rRNA product synthesized may not contain
sufficient biotin molecules to facilitate its removal by the
streptavidin microspheres.
[0155] Thus, the question arose as to whether the 35% biotin-UTP
used in Example 2 was the optimal concentration to ensure that all
antisense rRNA synthesized contained sufficient biotin molecules
for its subsequent removal by the streptavidin beads. To answer
this question, the following experiment tested biotinylated
antisense rRNAs made as described in Example 1 using either 50% or
60% biotin-UTP solutions. The standard 4-.mu.l mixture of the
respective biotinylated antisense 16S and 23S rRNA synthesized
using the 50% and 60% levels of biotin-UTP was then made. The
standard 4 .mu.l-mixture of the 35% biotinylated antisense rRNA
included as a control.
[0156] Three sets of two reactions, each comprising 4 .mu.l of the
35% (reactions #1 and #2) or 50% (reactions #3 and #4) or 60%
(reactions #5 and #6) biotinylated antisense rRNA mixture were
prepared in a final volume of 20 .mu.l in 1.times. hybridization
buffer. The reactions were incubated at 68.degree. C. for 5 minutes
and then at room temperature for 15 minutes. Reactions # 2, #4 and
#6 were each transferred to a fresh 1.5-ml microcentrifuge tube
containing 40 .mu.l of washed streptavidin microspheres and
reactions # 1, #3 and #5 were each transferred to a fresh 1.5-ml
microcentrifuge tube containing 40 .mu.l of bind/wash buffer. All
reactions were further incubated at room temperature for 15 minutes
with occasional gentle mixing (3-4 minutes) in order to allow
formation of the biotin-streptavidin complex. The reactions were
spun at 12,000 rpm for 5 minutes on a benchtop centrifuge and the
supernatant transferred to fresh 1.5-ml microcentrifuge tubes. Any
biotinylated antisense rRNA remaining in the supernatants were
purified using the ZYMO RNA Clean-up procedure and eluted in 13.5
.mu.l RNase-free water. Fifty percent of each purified sample was
analyzed by ethidium bromide-stained agarose gel electrophoresis as
shown in FIG. 3B.
[0157] FIG. 3B, Lanes 1, 3 and 5 show the biotinylated antisense
rRNA remaining in the absence of any microspheres for the 35%, 50%
and 60% biotin-UTP samples, respectively. With the addition of 40
.mu.l of streptavidin microspheres, there was no biotinylated
antisense rRNA visibly remaining in any of the corresponding
reactions (Lanes 2, 4 and 6). It was not possible to observe any
differences in the performance of the 35%, 50% and 60% biotinylated
antisense rRNA samples following treatment with 40 .mu.l of the
streptavidin microspheres by agarose gel analysis.
[0158] In order to further determine if there was a difference
between the use of the 35%, 50% and 60% biotinylated antisense
rRNA, the remaining half of each reaction was converted to
first-strand cDNA in a standard reverse transcriptase reaction
containing random hexamers and purified using the Qiaquick PCR
purification kit as recommended by the manufacturer (Qiagen). A
5-.mu.l aliquot of each was then used in separate PCR amplification
reactions containing primers specific to the terminal 5' and 3'
regions of 23S (SEQ ID NO: 15: 5'-GACGTGCTAATCTGCGATAAGC, SEQ ID
NO: 16: 5'-ATGGATTCAGTTAATGATAGTGTGTCG, SEQ ID NO: 17:
5'-CTGAAAGCATCTAAGCACGAAACTTGC and SEQ ID NO: 18:
5'-CCTATCAACGTCGTCGTCTTCAAC) and 16S (SEQ ID NO: 19:
5'-GCCTAACACATGCAAGTCGAAC, SEQ ID NO: 20:
5'-AGCTACCGTTTCCAGTAGTTATCC, SEQ ID NO: 21:
5'-CGGAATCGCTAGTAATCGTGGAT and SEQ ID NO: 22:
5'-TCCCGAAGGTTAAGCTACCTACTT) rRNAs for 20 PCR cycles. PCR
amplification is notably more sensitive than ethidium bromide
agarose gel analysis and should therefore better demonstrate any
differences between the removal efficiencies of the 35%, 50% and
60% biotinylated antisense rRNA by the microspheres. The results
are shown in FIG. 3C.
[0159] FIG. 3C, Lanes 1, 3 and 5 show the expected PCR amplicons
for the 5' 23S rRNA (Panel 1), 3' 23S rRNA (Panel 2), 5' 16S rRNA
(Panel 3) and 3' 16S rRNA (Panel 4) with the 35%, 50% and 60%
biotinylated antisense rRNA mixtures, respectively. Lanes 2, 4 and
6 show the corresponding PCR reaction products following the use of
the streptavidin microspheres. As seen in Lane 2 (Panels 2 and 4),
residual carryover of 35% biotinylated antisense rRNA resulted in
still some specific PCR-amplified products that were not observed
with the 50% and 60% biotinylated antisense rRNA materials. Both
the 50% and 60% biotinylated antisense rRNA materials behaved
similarly and thus, the 50% condition was selected.
Example 4
Removal of the 23S and 16S rRNA from Another Prokaryotic Species
(Lactobacillus plantarum) Using the E. coli Biotinylated Antisense
23S and 16S rRNA mixture
[0160] In order to test if the E. coli biotinylated antisense rRNA
mixture would be capable of removing the 23S and 16S rRNA sequences
exhibited by a relatively diverse prokaryotic species, total RNA
from Lactobacillus plantarum, which shares approximately 80%
homology with E. coli 16S and 23S rRNA was used. In one reaction,
500 ng of L. plantarum total RNA was mixed with 4 .mu.l of the E.
coli biotinylated antisense rRNA mixture described in Example 2
above in 1.times. hybridization buffer in a final volume of 20
.mu.l. A second reaction to serve as a control contained everything
as the first reaction minus the E. coli biotinylated antisense rRNA
mixture. The reactions were incubated at 68.degree. C. for 10
minutes and then at room temperature for 15 minutes. Each reaction
was then added to a fresh 1.5-ml microcentrifuge tube containing 50
.mu.l of washed and resuspended streptavidin-coated microspheres.
The reactions were further incubated at room temperature for 15
minutes with occasional gentle mixing (3-4 minutes) in order to
allow for formation of the biotin-streptavidin complex. The
reactions were then spun at 12,000 rpm for 5 minutes using a
benchtop centrifuge to pellet the microspheres, the supernatant
removed and the RNA purified using the ZYMO RNA Clean-up procedure.
Each RNA sample was eluted in 13.5 .mu.l RNase-free water and 50%
of each analyzed by ethidium bromide stained agarose gel
electrophoresis as shown FIG. 4.
[0161] FIG. 4, Lane 1 compared to Lane 2 shows that both the 16S
and 23S rRNA bands for L. plantarum were no longer visible
following subtraction with the E. coli biotinylated antisense rRNA
mixture indicating that they were efficiently subtracted as well
even though the rRNA sequences were only 80% homologous.
Example 5
Removal of 23S, 16S and 5S rRNA from a Range of Inputs of E. coli
Total RNA Using the E. coli Biotinylated Antisense rRNA Mixture
[0162] An E. coli biotinylated antisense rRNA mixture was prepared,
now containing 23S, 16S and 5S antisense rRNA, and the following
reactions for generating rRNA-depleted samples and control
reactions were set up as shown in Table 1, and were performed in
duplicate in a final volume of 20 .mu.l in 1.times. hybridization
buffer:
TABLE-US-00003 TABLE 1 E. coli biotinylated Streptavidin Reaction
E. coli total antisense rRNA mixture coated # RNA Inputs (23S, 16S,
5S) microspheres 1, 2 50 ng 2 .mu.l 25 .mu.l 3, 4 100 ng 2 .mu.l 25
.mu.l 5, 6 500 ng 4 .mu.l 50 .mu.l 7, 8 1000 ng 4 .mu.l 50 .mu.l 9,
10 2500 ng 8 .mu.l 100 .mu.l 11, 12 5000 ng 10 .mu.l 125 .mu.l 13,
14 500 ng -- 50 .mu.l
The reactions were incubated at 68.degree. C. for 10 minutes and
then at room temperature for 15 minutes. Each reaction was then
added to a fresh 1.5-ml microcentrifuge tube containing the
appropriate quantity of washed and resuspended streptavidin-coated
microspheres. The reactions were further incubated at room
temperature for 15 minutes with occasional gentle mixing (3-4
minutes) in order to allow for formation of the biotin-streptavidin
complex. The reactions were then spun at 12,000 rpm for 5 minutes
using a benchtop centrifuge to pellet the microspheres and the
supernatant removed and the RNA purified using the ZYMO RNA
Clean-up procedure. Each RNA sample was eluted in 13.5 .mu.l
RNase-free water. The entire RNA sample for each was then converted
to first-strand cDNA in a standard reverse transcriptase reaction
containing random hexamers and purified using the Qiaquick PCR
purification kit as recommended by the manufacturer (Qiagen). A
5-.mu.l aliquot of each first-strand cDNA sample was used in
separate PCR amplification reactions containing primers specific to
the terminal 5' regions of 23S (SEQ ID NO: 15 and SEQ ID NO: 16)
and 16S rRNA (SEQ ID NO: 19 and SEQ ID NO: 20), the complete 5S
rRNA (SEQ ID NO: 13 and SEQ ID NO: 23: 5'-TGCCTGGCAGTTCCCTACTCTC)
and the ompA mRNA (SEQ ID NO: 24: 5'-ACCAGGTTAACCCGTATGTTGGCT and
SEQ ID NO: 25: 5'-ACCGATGTTGTTGGTCCACTGGTA) for 20 cycles. A
control reaction minus template was also performed for each primer
pair. A 5-.mu.l aliquot of each PCR reaction was analyzed by
ethidium bromide stained agarose gel electrophoresis as shown in
FIG. 5.
[0163] FIG. 5, Lanes 1-12 show excellent reduction in the amount of
23S (Panel A), 16S (Panel B) and 5S (Panel C) rRNA sequences
contained in the different inputs of E. coli total RNA in
duplicate. Whereas, the ompA mRNA (Panel D) transcripts was still
clearly detected in all samples and appeared to be unperturbed
compared to the corresponding non-subtracted samples (Lanes 7 and 8
versus Lane 13 and 14). Clearly, this method for rRNA subtraction
appears to be applicable across a broad range of total RNA inputs
with at least a 100-fold dynamic range in this example.
Example 6
Subtraction of Fragmented E. coli 23S, 16S and 5S rRNA Sequences
Using the E. coli Biotinylated Antisense rRNA Mixture
[0164] Ten micrograms of E. coli total RNA was fragmented in
1.times. fragmentation buffer (Ambion) at 65.degree. C. for 3
minutes. The fragmented RNA was purified using the ZYMO RNA
Clean-up procedure, eluted in 20 .mu.l RNase-free water and the
concentration determined by measuring the absorbance at 260 nm with
a spectrophotometer. A 300 ng aliquot of the fragmented RNA
compared to the intact RNA was analyzed by ethidium bromide stained
agarose gel electrophoresis as shown in FIG. 6A.
[0165] FIG. 6A, Lane 1 shows the intact E. coli total RNA and Lane
2 shows the corresponding fragmented total RNA. The intact 23S and
16S rRNA bands were no longer visible in the fragmented sample
contained in Lane 2.
[0166] Next, ribosomal RNA subtraction was applied to a 2.5 .mu.g
sample of the fragmented E. coli total RNA sample comparing it to a
similar amount of intact E. coli total RNA. The following four
reactions were assembled--reactions #1 and #2 contained 2.5 .mu.g
of intact E. coli total RNA each and reactions #3 and #4 contained
2.5 .mu.g of the fragmented E. coli total RNA each. Next, to each
of reactions #2 and #4, 8 .mu.l of the E. coli biotinylated
antisense rRNA mixture was added and the final reaction volume
adjusted to 40 .mu.l in 1.times. hybridization buffer. The
reactions were incubated at 68.degree. C. for 10 minutes and then
at room temperature for 15 minutes. Each reaction was transferred
to a fresh 1.5-ml microcentrifuge tube containing 50 .mu.l of
washed streptavidin microspheres. The reactions were further
incubated at room temperature for 15 minutes with occasional gentle
mixing (3-4 minutes) in order to allow for formation of the
biotin-streptavidin complex. The reactions were spun at 12,000 rpm
for 5 minutes on a benchtop centrifuge and each supernatant
transferred to a fresh 1.5-ml microcentrifuge tube. The RNA
contained in the supernatants were purified using the ZYMO RNA
Clean-up procedure and eluted in 13.5 .mu.l RNase-free water. Fifty
percent of each purified sample was analyzed by ethidium bromide
stained agarose gel electrophoresis and Northern blot analysis as
shown in FIG. 6B.
[0167] FIG. 6B, Panel 1 (Lanes 2 and 4) show the RNA remaining
following rRNA subtraction. In the case of the intact total RNA, it
is clearly evident that the 23S and 16S rRNA bands are removed
(Lane 2 versus Lane 1). Whereas, for the fragmented total RNA,
there is an overall reduction in the amount of RNA following
subtraction as would be expected if the fragments of rRNA were also
subtracted (Lanes 4 versus Lane 3). The Northern blot analysis for
ompA mRNA (Panel 2) and 23S (Panel 3), 16S (Panel 4) and 5S (Panel
5) rRNA clearly show similar and excellent subtraction of the
different rRNA sequences in both the intact total RNA (Panel 3, 4,
and 5--Lane 2) and fragmented total RNA (Panel 3, 4, and 5--Lane 4)
compared to the respective non-subtracted samples (Panel 3, 4, and
5--Lane 1 and 3). At the same time, the amount of ompA mRNA
sequence appears to be unperturbed by the subtraction process for
both the intact total RNA (Panel 2--Lane 2 versus Lane 1) and the
fragmented total RNA (Panel 2--Lane 4 versus Lane 3).
[0168] In addition, the remaining 50% of each purified RNA sample
was converted to first-strand cDNA in a standard reverse
transcriptase reaction containing random hexamers and purified
using the Qiaquick PCR purification kit as recommended by the
manufacturer (Qiagen). A 5-.mu.l aliquot of each was then used in
separate PCR amplification reactions containing primers specific to
the terminal 5' regions of 23S (SEQ ID NO: 15 and SEQ ID NO: 16)
and 16S rRNA (SEQ ID NO: 19 and SEQ ID NO: 20), the complete 5S
rRNA (SEQ ID NO: 13 and SEQ ID NO: 23) and the ompA mRNA (SEQ ID
NO: 24 and SEQ ID NO: 25) for 20 cycles. A 5-.mu.l aliquot of each
PCR reaction was analyzed by ethidium bromide stained agarose gel
electrophoresis as shown in FIG. 6C.
[0169] FIG. 6C shows that by RT-PCR analysis, there was excellent
subtraction of 23S (Panel 2), 16S (Panel 3) and 5S rRNA (Panel 4)
for both the intact (Lane 2 versus Lane 1) and the fragmented (Lane
4 versus Lane 3) E. coli total RNA. Whereas, the ompA mRNA was
equally preserved in both cases before and after subtraction (Panel
1).
Example 7
Specific Hybridization of Human, Mouse and Rat 28S and 18S rRNA to
Human Biotinylated Antisense 28S and 18S rRNA, and Their Removal
with Streptavidin Microspheres
[0170] Intact total RNA (500 ng) samples for human (reactions #4
and #5), mouse (reactions #7 and #8) and rat (reactions #10 and
#11) were mixed with either 2.1 .mu.g of biotinylated antisense 28S
rRNA or 1.0 .mu.g of biotinylated antisense 18S rRNA in 1.times.
hybridization buffer in a final volume of 20 .mu.l. Corresponding
intact total RNA samples (reactions #3, #6 and #9) and biotinylated
antisense rRNA alone (#1 and #2) were included as controls. All
reactions were incubated at 68.degree. C. for 10 minutes followed
by room temperature for 15 minutes. The RNA contained in each
sample was then purified using the ZYMO RNA Clean-up procedure and
eluted in 13.5 .mu.l RNase-free water. The entire sample for each
was analyzed by ethidium bromide stained agarose gel
electrophoresis as shown in FIG. 7A.
[0171] FIG. 7A, Lanes 1 and 2 show the individual biotinylated
antisense 23S and 18S rRNA, respectively. Lanes 3, 6 and 9 show the
individual intact total RNA corresponding to human, mouse and rat,
respectively. Lanes 4, 7 and 10 show the human, mouse and rat total
RNA samples, respectively following hybridization to the
biotinylated antisense 28S rRNA. Clearly, the 28S rRNA band in the
different total RNA samples have hybridized to its complementary
antisense rRNA since it now migrates with a high molecular weight
higher whereas, the 18S rRNA remained unperturbed. When the
different total RNA samples were then hybridized to the
biotinylated antisense 18S rRNA, clearly the 18S rRNA band now
migrated with a higher molecular weight (Lane 5, 8 and 11) and now,
the 28S band was unperturbed. It's evident from these results that
each biotinylated antisense rRNA was hybridizing specifically to
its complementary target and equally well for human, mouse and rat
that share at least 99% sequence homology.
[0172] Next, a biotinylated antisense rRNA mixture for human
comprising approximately 1.2 pmoles each of 28S and 18S antisense
rRNA in a final volume of 4 .mu.l of RNase-free water was prepared
using the in vitro synthesized RNA made as described in Example 1.
Intact total RNA (2.5 .mu.g) from human (HeLa), mouse (3T3) and rat
(NRK) were each mixed with 8 .mu.l of the biotinylated antisense
rRNA solution in 1.times. hybridization buffer in a final volume of
40 .mu.l. A control reaction containing only 2.5 .mu.g of the
corresponding intact total RNA was also included. The reactions
were incubated at 68.degree. C. for 10 minutes followed by room
temperature for 15 minutes. Each reaction was transferred to a
fresh 1.5-ml microcentrifuge tube containing 100 .mu.l of washed
streptavidin microspheres, respectively. The reactions were further
incubated at room temperature for 15 minutes with occasional gentle
mixing (3-4 minutes) in order to allow formation of the
biotin-streptavidin complex and then 37.degree. C. for 5 minutes.
The reactions were spun at 14,000 rpm for 5 minutes on a benchtop
centrifuge and each supernatant transferred to a fresh 1.5-ml
microcentrifuge tube. The RNA contained in the supernatants were
purified using the ZYMO RNA Clean-up procedure and eluted in 13.5
.mu.l RNase-free water. Fifty percent of each purified RNA sample
was analyzed by ethidium bromide stained agarose gel
electrophoresis as shown in FIG. 7B.
[0173] FIG. 7B, Lanes 1, 3 and 5 show the individual total RNA
without any 28S and 18S rRNA subtraction, whereas, Lanes 2, 4, and
6 show the corresponding RNA remaining following 28S and 18S rRNA
subtraction. It is clear from these results that a majority of the
28S and 18S rRNA bands were removed following the subtraction
procedure as described.
Example 8
Subtraction of 28S, 18S, 5.8S and 5S rRNA from Varying Amounts of
intact human Total RNA Using a Human Biotinylated Antisense rRNA
Mixture
[0174] A biotinylated antisense rRNA mixture for human comprising
approximately 1.2 pmoles each of 28S, 18S, 5.8S and 5S antisense
rRNA in a final volume of 4 .mu.l of RNase-free water was prepared
using the in vitro synthesized RNA made as described in Example 1.
Ribosomal RNA subtraction reactions comprising 100 ng (reaction
#1), 500 ng (reaction #2) and 5.0 .mu.g (reaction #3) of intact
HeLa total RNA with 2 .mu.l, 4 .mu.l and 8 .mu.l of the
biotinylated human antisense rRNA mixture, respectively were
prepared in 1.times. hybridization buffer in a final volume of 20
.mu.l. A control reaction (reaction #4) with only 500 ng of the
intact HeLa total RNA was also included. The reactions were
incubated at 68.degree. C. for 10 minutes followed by room
temperature for 15 minutes. Each reaction was transferred to a
fresh 1.5-ml microcentrifuge tube containing 25 .mu.l, 50 .mu.l,
125 .mu.l and 50 .mu.l of washed streptavidin microspheres,
respectively. The reactions were further incubated at room
temperature for 15 minutes with occasional gentle mixing (3-4
minutes) in order to allow formation of the biotin-streptavidin
complex and then 37.degree. C. for 5 minutes. The reactions were
spun at 14,000 rpm for 5 minutes on a benchtop centrifuge and each
supernatant transferred to a fresh 1.5-ml microcentrifuge tube. The
RNA contained in the supernatants were purified using the ZYMO RNA
Clean-up procedure and eluted in 13.5 .mu.l RNase-free water. Next,
each purified RNA sample was converted to first-strand cDNA in a
standard reverse transcriptase reaction containing random hexamers
and purified using the Qiaquick PCR purification kit as recommended
by the manufacturer (Qiagen). A 5-.mu.l aliquot of each was then
used in separate PCR amplification reactions containing primers
specific to the terminal 5' and 3' regions of 28S (SEQ ID NO: 26:
5'-CTCAGTAACGGCGAGTGAAC, SEQ ID NO: 27: 5'-GCCTCGATCAGAAGGACTTG,
SEQ ID NO: 28: 5'-TACCACAGGGA TAACTGGCT and SEQ ID NO: 29:
5'-TAGGAAGAGCCGACATCGAA) and 18S rRNA (SEQ ID NO: 30:
5'-CCTACCTGGTTGATCCTGCC, SEQ ID NO: 31: 5'-CCAAGTAGGAGAGGAGCGAG,
SEQ. ID. No. 32: 5'-CCCAGTAAGTGC GGGTCATA and SEQ ID NO: 33:
5'-TCACTAAACCATCCAATCGGTAGTA) the complete 5.8S rRNA (SEQ ID NO: 3
and SEQ ID NO: 34: 5'-GATCCTTCCGCAGGT TCACCTAC), the complete 5S
rRNA (SEQ ID NO: 5 and SEQ ID NO: 35: 5'-AAGCCTACAGCACCCGGTATTC)
and 5' GAPDH mRNA (SEQ ID NO: 36: 5'-TCGACAGTCAGCCGCATCTTCTTT and
SEQ ID NO: 37: 5'-ACCAAATCCGTTG ACTCCGACCTT) for 20 cycles. A
control reaction for each primer pair minus template was included.
A 5-.mu.l aliquot of each PCR reaction was analyzed by ethidium
bromide stained agarose gel electrophoresis shown in FIG. 8.
[0175] FIG. 8, Lanes 1, 2 and 3 show the RT-PCR results for 28S
rRNA (Panel A), 18S rRNA (Panel B), 5.8S rRNA (Panel C), 5S rRNA
(Panel D) and GAPDH mRNA (Panel E) for the 100 ng, 500 ng and 5.0
.mu.g inputs of total RNA subtracted with the biotinylated human
antisense rRNA mixture, respectively. Lane 4 shows the PCR for the
500 ng non-subtracted total RNA input. It is evident from the
results that there is excellent subtraction over a broad range of
total RNA inputs for the different ribosomal RNA sequences using
the biotinylated antisense rRNA mixture (Lanes 1, 2 and 3 versus
Lane 4).
Example 9
Subtraction of Human rRNA Sequences Exhibited by Various Levels of
Fragmented Human Total RNA
[0176] HeLa intact total RNA was fragmented in 1.times.
fragmentation buffer (Ambion) for 1, 2 and 3 minutes. The RNA
samples were purified using the ZYMO RNA Clean-up procedure, eluted
RNase-free water and the concentration determined at an absorbance
of 260 nm using a spectrophotometer.
[0177] Duplicate reactions comprising either 2.5 .mu.g of the
intact (reactions # and #2) or fragmented (reactions #3, #4, #5,
#6, #7 and #8) total RNA samples were prepared in 1.times.
hybridization buffer. To one of each duplicate reaction (#2, #4, #6
and #8), 8 .mu.l of the biotinylated human antisense rRNA mixture
was added and the final volume for each reaction adjusted to 40
.mu.l. The reactions were incubated at 68.degree. C. for 10 minutes
followed by room temperature for 15 minutes. Each reaction was
transferred to a fresh 1.5-ml microcentrifuge tube containing 100
.mu.l of washed streptavidin microspheres. The reactions were
further incubated at room temperature for 15 minutes with
occasional gentle mixing (3-4 minutes) in order to allow formation
of the biotin-streptavidin complex and then 37.degree. C. for 5
minutes. The reactions were spun at 14,000 rpm for 5 minutes on a
benchtop centrifuge and each supernatant transferred to a fresh
1.5-ml microcentrifuge tube. The RNA contained in the supernatants
were purified using the ZYMO RNA Clean-up procedure and eluted in
13.5 .mu.l RNase-free water. Fifty percent of each purified RNA
sample was analyzed by ethidium bromide stained gel electrophoresis
as shown in FIG. 9A.
[0178] FIG. 9A, Lanes 4, 6 and 8, containing the fragmented total
RNA and biotinylated antisense rRNA show a significant reduction in
the amount of RNA as a result of subtraction compared to the
respective controls (Lanes 3, 5 and 7). In Lane 2 compared to Lane
1 with the intact total RNA, the full-length rRNA bands were no
longer visible.
[0179] Next, the remaining 50% of each purified RNA sample was
converted to first-strand cDNA in a standard reverse transcriptase
reaction containing random hexamers and purified using the Qiaquick
PCR purification kit as recommended by the manufacturer (Qiagen). A
5-.mu.l aliquot of each was then used in separate PCR amplification
reactions containing primers specific to the terminal 5' regions of
28S (SEQ ID NO: 26 and SEQ ID NO: 27) and 18S rRNA (SEQ ID NO: 30
and SEQ ID NO: 31), the complete 5.8S rRNA (SEQ ID NO: 3 and SEQ ID
NO: 34), the complete 5S rRNA (SEQ ID NO: 5 and SEQ ID NO: 35) and
5' GAPDH mRNA (SEQ ID NO: 36 and SEQ ID NO: 37) for 20 cycles. A
control reaction minus template was also performed for each primer
pair. A 5-.mu.l aliquot of each PCR reaction was analyzed by
ethidium bromide stained agarose gel electrophoresis shown in FIG.
9B.
[0180] FIG. 9B, Lanes 2, 4, 6 and 8 show the PCR results for 28S
rRNA (Panel 1), 18S rRNA (Panel 2), 5.8S rRNA (Panel 3), 5S rRNA
(Panel 4) and GAPDH mRNA (Panel 5) for the intact, and fragmented
(1, 2 and 3 minutes) total RNA, respectively following subtraction.
The corresponding non-subtracted control reactions are shown in
Lanes 1, 3, 5 and 7. It is evident from the results that there is
excellent subtraction over a broad range of fragmented total RNA
inputs similar to the intact total RNA for the different ribosomal
RNA sequences using the biotinylated antisense rRNA mixture.
Example 10
Comparison of E. coli rRNA Subtraction Using the Method of the
Present Invention and the MICROBExpress.TM. Method
[0181] A kit that uses conserved rRNA (23S and 16S) oligonucleotide
sequences to subtract the corresponding rRNA from inputs of 2
.mu.g-10 .mu.g from prokaryotic total RNA was purchased from Ambion
for comparison to the exemplary methods in these Examples. An 15
.mu.g aliquot of the intact control E. coli total RNA supplied in
the MICROBExpress.TM. kit was first fragmented in 1.times.
fragmentation buffer (Ambion) at 65.degree. C. for 2 minutes,
purified using the ZYMO RNA Clean-up procedure, eluted RNase-free
water and the concentration determined at an absorbance of 260 nm
using a spectrophotometer. An equal aliquot (2.5 .mu.g) of either
the intact or fragmented E. coli total RNA was then used in
subtraction reactions following either the exemplary method
described in Example 9 ("Exemplary Method") for the appropriate RNA
samples or the MICROBExpress.TM. method exactly as described by the
manufacturer (Ambion). Control reactions representing no rRNA
subtraction were included for both methods. Each RNA sample was
then purified by EtOH precipitation as described for the
MICROBExpress.TM. procedure and resuspended in 12 .mu.l RNase-free
water. Fifty percent of each purified RNA sample was analyzed by
ethidium bromide stained agarose gel electrophoresis and Northern
blot as shown in FIG. 10A.
[0182] FIG. 10A, Lanes 1, 2 and 3, 4 show the relative rRNA
subtraction from intact and fragmented total RNA using the
Exemplary Method and the MICROBExpress.TM. method for 23S rRNA
(Panel 2), 16S rRNA (Panel 3) and 5S rRNA (Panel 4). Clearly, for
both the intact and fragmented total RNA samples, the different
rRNA sequences were subtracted significantly better using the
Exemplary Method invention (Exemplary Method--Lanes 2 and 4)
compared to the MICROBExpress method (MICROBExpress.TM.
Panel--Lanes 2 and 4). It appears that even the control intact RNA
supplied with the MICROBExpress.TM. contain a level of fragmented
rRNA sequences that were clearly not removed by the MicrobExpress
method (MICROBExpress.TM. Panel--Lane 2) but were clearly removed
using the Exemplary Method described herein (Exemplary Method,
Panel--Lane 2). In addition, Panel 1 shows that the ompA mRNA
sequence remain unperturbed before and after subtraction for both
methods.
[0183] Next, the remaining 50% of each purified RNA sample was
converted to first-strand cDNA in a standard reverse transcriptase
reaction containing random hexamers and purified using the Qiaquick
PCR purification kit as recommended by the manufacturer (Qiagen). A
5-.mu.l aliquot of each was then used in separate PCR amplification
reactions containing primers specific to the terminal 5' region of
23S (SEQ ID NO: 15 and SEQ ID NO: 16) and 16S rRNA (SEQ ID NO: 19
and SEQ ID NO: 20) the complete 5S rRNA (SEQ ID NO: 13 and SEQ ID
NO: 23) and ompA mRNA (SEQ ID NO: 24 and SEQ ID NO: 25) for 20
cycles. A 5-.mu.l aliquot of each PCR reaction was analyzed by
ethidium bromide stained agarose gel electrophoresis shown in FIG.
10B.
[0184] FIG. 10B, Lanes 2 and 4 show the PCR results for 23S rRNA
(Panel 2), 16S rRNA (Panel 3), 5S rRNA (Panel 4) and ompA mRNA
(Panel 1) for the intact and fragmented E. coli total RNA,
respectively following subtraction using the Exemplar Method and
the MICROBExpress.TM. method. The corresponding non-subtracted
control reactions are shown in Lanes 1 and 3. It is evident from
the results that there is excellent subtraction of the different
rRNA sequences exhibited by both the intact and fragmented total
RNA samples using the Exemplary Method (Exemplary Method Panel)
compared to the MICROBExpress.TM. method (MicroExpress Panel). In
both cases though, the ompA mRNA was similarly maintained.
Example 11
Comparison of Human rRNA Subtraction Using an Exemplary Method vs.
the OLIGOTEX Poly A+ mRNA Purification Method
[0185] A kit that uses oligonucleotide dT to isolate mRNA from
eukaryotic total RNA was purchased from Qiagen for comparison to
the Exemplary Method described in Example 9 above. When fragmented
RNA was used, the RNA was first fragmented in 1.times.
fragmentation buffer (Ambion) at 65.degree. C. for 2 minutes. The
intact or fragmented RNA was purified using the ZYMO RNA Clean-up
procedure, eluted RNase-free water and the concentration determined
at an absorbance of 260 nm using a spectrophotometer. An equal
aliquot (2.5 .mu.g) of either the intact or fragmented E. coli
total RNA was then used in rRNA subtraction reactions using the
Exemplary Method for the appropriate RNA samples or the Oligotex
poly A+ mRNA purification method exactly as described by the
manufacturer (Qiagen). Control reactions representing no rRNA
subtraction were included for both methods. Each RNA sample was
then purified by ethanol precipitation and resuspended in 12 .mu.l
RNase-free water. Fifty percent of each purified RNA sample was
analyzed by ethidium bromide stained agarose gel electrophoresis as
shown in FIG. 11A.
[0186] FIG. 11A, Lanes 1, 2 and 3, 4 show the relative rRNA removal
for intact and fragmented total RNA using the Exemplary Method and
the OLIGOTEX poly A+ mRNA purification method. Clearly, for both
the intact and fragmented total RNA samples, the different rRNA
sequences were visibly removed using the Exemplary Method (Panel
1--Lanes 2 and 4) and the OLIGOTEX method (Panel 2--Lanes 2 and 4)
compared to the respective controls (Panel 1--Lanes 1 and 3 and
Panel 2--Lanes 1 and 3). In addition, it appears that there was
very little mRNA visibly remaining following the OLIGOTEX method
and all of the types of small RNA molecules (tRNA, miRNA etc.)
appear to have been eliminated as well (Panel 2--Lanes 2 and
4).
[0187] Next, the remaining 50% of each purified RNA sample was
converted to first-strand cDNA in a standard reverse transcriptase
reaction containing random hexamers and purified using the Qiaquick
PCR purification kit as recommended by the manufacturer (Qiagen). A
5-.mu.l aliquot of each was then used in separate PCR amplification
reactions containing primers specific to the terminal 5' and 3'
regions of 28S (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ
ID NO: 29) and 18S rRNA (SEQ SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 32 and SEQ ID NO: 33), the complete 5.8S rRNA (SEQ ID NO: 3 and
SEQ ID NO: 34) and the complete 5S rRNA (SEQ ID NO: 5 and SEQ ID
NO: 35) for 20 cycles. A 5-.mu.l aliquot of each PCR reaction was
analyzed by ethidium bromide stained agarose gel electrophoresis
shown in FIG. 11B.
[0188] FIG. 11B, Lanes 2 and 4 show the PCR results for 28S rRNA
(Panels 1 and 2), 18S rRNA (Panel 3 and 4), 5.8S rRNA (Panel 5) and
5S rRNA (Panel 6) for the intact and fragmented HeLa total RNA,
respectively following rRNA subtraction using the Exemplary Method
and the OLIGOTEX method. The corresponding non-subtracted control
reactions are shown in Lanes 1 and 3. It is evident from the
results that there is overall excellent subtraction of the
different rRNA sequences exhibited by both the intact and
fragmented total RNA samples using the Exemplary Method (Exemplary
Method Panel) and to the OLIGOTEX method (OLIGOTEX Panel). In fact,
the Exemplary Method appears to be somewhat better than the
OLIGOTEX method for both the 28S and 18S rRNA sequences (Panels
1-4) whereas, for the 5.8S and 5S rRNA sequences, the OLIGOTEX
method appears to be slightly better (Panel E and F,
respectively).
[0189] In addition, PCR primers specific for the terminal 5' and 3'
regions of GAPDH (SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38:
5'-CACAAGAGGAAGAGAGAGA CCCTCA and SEQ ID NO: 39:
5'-TTGATGGTACATGACAAGGTGCGG) and PGK1 (SEQ ID NO: 40:
5'-GAATCACCGACCTCTCTCCC, SEQ. ID. No. 41: 5'-CGACTCTCATAACGACCCGC,
SEQ ID NO: 42: 5'-CCAGAGGTGACCACTTTCAA and SEQ ID NO: 43:
5'-ATGTGGAACAGAGCCTTCCTC) mRNA were used in PCR (GAPDH: 22 cycles
and PGK1: 26 cycles) with the random primed cDNA templates and a
5-.mu.l aliquot of each PCR reaction was analyzed by ethidium
bromide stained agarose gel electrophoresis as shown in FIG. 11C.
It is clearly evident from the PCR results that there is a
reduction in the amount of mRNA for both GAPDH (Panels 1 and 2) and
PGK 1 (Panels 3 and 4) in the OLIGOTEX method for both intact and
fragmented total RNA (OLIGOTEX Panel, Lanes 2 and 4, respectively)
compared to the non-rRNA-subtracted samples (OLIGOTEX Panel, Lanes
1 and 3, respectively), using the Exemplary Method (Exemplary
Method Panel, Lanes 2 and 4). Furthermore, the Exemplary Method did
not show any significant loss in the same mRNA sequences for both
intact and fragmented total RNA (Exemplary Method Panel, Lanes 2
and 4, respectively) compared to the non-rRNA subtracted samples
(Exemplary Method Panel, Lanes 1 and 3, respectively). In addition,
the 5' regions of both GAPDH (Panel 1, Lane 4) and PGK1 (Panel 3,
Lane 4) for the fragmented total RNA sample were lost with the
OLIGOTEX method (OLigotex Panel) since the poly A tail was no
longer present but not so for the Exemplary Method (Exemplary
Method Panel, Lane 4 (Panel 1 and 3), respectively). Overall, these
results demonstrate a clear benefit of the Exemplary Method
compared to the OLIGOTEX method.
[0190] Next, PCR primers specific for Poly A- and bimorphic
transcripts (SEQ ID NO: 44: 5'-CACGTTTTCTCAGCTGCTTG and SEQ ID NO:
45: 5'-TTCACCTTTTCATC CAAGGC for transcript #1, SEQ ID NO: 46:
5'-GTGTGGTGGTGTGTGCCTAT and SEQ ID NO: 47: 5'-GAGACATGGTCTTGCTCCGT
for transcript #3, SEQ ID NO: 48: 5'-TAGCTCAGTGGTAGAGCGCA and SEQ
ID NO: 49: 5'-GATTTGCTCAGCA GCACGTA for transcript #15 and SEQ ID
NO: 50: 5'-CACTTGGGGACACTTTCCAG and SEQ ID NO: 51:
5'-TCAGGGAAAATGAGCCAATC for bimorphic transcript #5 (Poly A-
Transcripts Expressed in HeLa Cells Qingfa Wu et. al. (July 2008).
PLoS One (www followed by
"plosone.org/article/info:doi/10.1371/journal.pone.0002803")) were
used in PCR with the random primed cDNA templates (36, 22, 28 and
38 cycles, respectively) and a 5-.mu.l aliquot of each PCR reaction
was analyzed by ethidium bromide stained agarose gel
electrophoresis as shown in FIG. 11D. It is clearly evident from
the PCR results that the poly A- and biomorphic transcripts were no
longer present following the OLIGOTEX mRNA purification method for
both intact and fragmented total RNA (OLIGOTEX Panel, Lanes 2 and
4, respectively) whereas, these sequences were maintained following
rRNA subtraction using the Exemplary Method for both intact and
fragmented total RNA (Exemplary Method PANEL, Lane 2 and 4,
respectively). These results point to another clear benefit of the
rRNA subtraction methods described in this application for
maintaining a broader spectrum of transcripts allowing for better
representation of the cellular content.
Example 12
Comparison of Human rRNA Subtraction Using an Exemplary Method of
the Present Invention vs. the RiboMinus.TM. Eukaryote Kit for
RNA-Seq
[0191] A kit (RiboMinus.TM. Eukaryote Kit for RNA-Seq) that uses
two conserved oligonucleotide sequences for each of 28S, 18S, 5.8S
and 5S rRNA to subtract the corresponding rRNA from inputs of 2
.mu.g--10 .mu.g from eukaryotic total RNA was purchased from
InVitrogen (Burlington, ON) for comparison to an examplary method
of the present invention. When fragmented total RNA was used, the
initial total RNA was first fragmented in 1.times. fragmentation
buffer (Ambion, Austin, Tex.) at 65.degree. C. for 2 minutes. The
intact or fragmented total RNA was purified using the ZYMO RNA
Clean-up procedure, eluted RNase-free water and the concentration
determined at an absorbance of 260 nm using a spectrophotometer. An
equal aliquot (2.5 .mu.g) of either the intact or fragmented HeLa
total RNA was then used in rRNA subtraction reactions using the
Exemplary Method for the appropriate RNA samples or the
RiboMinus.TM. Eukaryote Kit for RNA-Seq method exactly as described
by the manufacturer (InVitrogen). Control reactions representing no
rRNA subtraction were included for both methods. Each RNA sample
was then purified by ethanol precipitation and resuspended in 12
.mu.l RNase-free water. Fifty percent of each purified RNA sample
was analyzed by ethidium bromide stained agarose gel
electrophoresis as shown in FIG. 12A.
[0192] FIG. 12A, Lanes 1, 2 and 3, 4 show the relative rRNA removal
for intact and fragmented total RNA using the Exemplary Method and
the RiboMinus.TM. Eukaryote Kit for RNA-Seq method. Clearly, for
both the intact and fragmented total RNA samples, the different
rRNA sequences were visibly removed using the Exemplary Method
(Exemplary method--Lanes 2 and 4) whereas for the RiboMinus.TM.
Eukaryote Kit for RNA-Seq method, only the intact total RNA showed
reduction of the rRNA sequences (RiboMinus.TM. method--Lanes 2)
compared to the respective non-rRNA-subtracted samples (Exemplary
method--Lanes 1 and 3 and RiboMinus.TM. method--Lane 1). There
appeared to be only minimal reduction of the rRNA-subtracted
fragmented total RNA sample using the RiboMinus.TM. Eukaryote Kit
for RNA-Seq as judged by the ethidium bromide stained intensity
(RiboMinus.TM. method--Lane 4) compared to the non-rRNA-subtracted
sample (RiboMinus.TM. method--Lane 3) likely due to the fact that
those ribosomal fragments that are not represented by the consensus
oligonucleotide sequences in the kit would therefore not be
removed.
[0193] Next, the remaining 50% of each purified RNA sample was
converted to first-strand cDNA in a standard reverse transcriptase
reaction containing random hexamers and purified using the Qiaquick
PCR purification kit as recommended by the manufacturer (Qiagen). A
5-.mu.l aliquot of each was then used in separate PCR amplification
reactions containing primers specific to the terminal 5' and 3'
regions of 28S (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ
ID NO: 29) and 18S rRNA (SEQ SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 32 and SEQ ID NO: 33), the complete 5.8S rRNA (SEQ ID NO: 3 and
SEQ ID NO: 34) and the complete 5S rRNA (SEQ ID NO: 5 and SEQ ID
NO: 35) for 20 cycles. A 5-.mu.l aliquot of each PCR reaction was
analyzed by ethidium bromide stained agarose gel electrophoresis
shown in FIG. 12B.
[0194] FIG. 12B, Lanes 2 and 4 show the PCR results for 28S rRNA
(Panels 1 and 2), 18S rRNA (Panel 3 and 4), 5.8S rRNA (Panel 5) and
5S rRNA (Panel 6) for the intact and fragmented HeLa total RNA,
respectively following rRNA subtraction using the Exemplary Method
and the RiboMinus.TM. Eukaryote Kit for RNA-Seq method. The
corresponding non-rRNA-subtracted control reactions are shown in
Lanes 1 and 3. It is evident from the results that there is overall
excellent subtraction of the 28S rRNA (Panels 1 and 2) and 18S rRNA
(Panel 3 and 4) sequences contained in both the intact and
fragmented total RNA samples using the Exemplary Method (Exemplary
Method Panel) but not the RiboMinus.TM. Eukaryote Kit for RNA-Seq
method (RiboMinus.TM. Panel). However, both methods showed similar
removal of the 5.8S (Panel 5) and 5S rRNA sequences (Panel 6).
[0195] In addition, PCR primers specific for the terminal 5' and 3'
regions of GAPDH (SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38:
5'-CACAAGAGGAAGAGAGAGA CCCTCA and SEQ ID NO: 39:
5'-TTGATGGTACATGACAAGGTGCGG) mRNA was used in PCR (GAPDH: 24
cycles) with the random primed cDNA templates and a 5-.mu.l aliquot
of each PCR reaction was analyzed by ethidium bromide stained
agarose gel electrophoresis as shown in FIG. 12C. It is clearly
evident from the PCR results that there is no obvious reduction in
the amount of mRNA for GAPDH (Panels 1 and 2) for both methods with
either the intact or fragmented HeLa total RNA (Lanes 2 and 4)
compared to the non-rRNA-subtracted samples (Lanes 1 and 3),
respectively. Overall, these results demonstrate a clear benefit of
the Exemplary Method in the removal of 18S and 28S rRNA sequences
independent of the state (intact or fragmented) of the total RNA
sample compared to the RiboMinus.TM. Eukaryote Kit for RNA-Seq
method. In fact, even for the intact total RNA sample, the
Exemplary method appeared to be more efficient at removal of 18S
and 28S rRNA sequences likely due, at least in part, to the fact
that there is invariably some degree of fragmented rRNA in even a
most intact total RNA sample that would not be removed by the
RiboMinus.TM. method. In fact, the RiboMinus.TM. Eukaryote Kit for
RNA-Seq method clearly states that this method requires use of only
high-quality total RNA samples.
[0196] Next, the random primed cDNA samples were analyzed by QPCR
as follows: The cDNA samples were diluted to anywhere from 2-fold
to 10-fold depending on the starting amount. Then, 1 .mu.l of each
dilution was added to a 25 .mu.l qPCR reaction comprising
1.times.FS PreMix E (GREEN), 12.5 pmole of forward and reverse PCR
primers and 1 unit of FS Enzyme Mix. The cycling conditions were
98.degree. C. for 2 minutes, followed by 40-45 cycles of 98.degree.
C. for 5 seconds, 60.degree. C. for 15 seconds and 72.degree. C.
for 30 seconds using the Bio-Rad iCycler (Bio-Rad, Hercules,
Calif.). The following QPCR primer pairs were used for 18S (SEQ.
ID. No. 30: SEQ. ID. No. 31, SEQ. ID. No. 52:
5'-CTTAGAGGGACAAGTGGCG, SEQ. ID. No. 53: 5'-GTAGGGTAGGCACACGCTGA,
SEQ. ID. No. 54: 5'-GAAACTTAAAGGAATTGACGGAAG, SEQ. ID. No. 55:
5'-GAATCGAGAAAGAGCTATCAATC, SEQ. ID. No. 56:
5'-CGATTGGATGGTTTAGTGAGG and SEQ. ID. No. 57:
5'-CCTTGTTACGACTTTTACTTCCTCTAG), 28S (SEQ. ID. No. 58:
5'-GCCGAAACGATCTCAACCTA, SEQ. ID. No. 59: 5'-CGCCAGTTCTGCTTACCAAA,
SEQ. ID. No. 60: 5'-CGGACCAAGGAGTCTAACA, SEQ. ID. No. 61:
5'-CAGGCATAGTTCACCATCTTTCG, SEQ. ID. No. 62:
5'-GGAGAGGGTGTAAATCTCGC, SEQ. ID. No. 63: 5'-GCCGACTTCCCTTACCTACA,
SEQ. ID. No. 64: 5'-GTGTCAGAAAAGTTACCACAGG, SEQ. ID. No. 65:
5'-GGCGAATTCTGCTTCACAATGATAG, SEQ. ID. No. 66:
5'-GGGAGTAACTATGACTCTCTTAAGGT, SEQ. ID. No. 67:
5'-TTGGCTGTGGTTTCGCTGGAT, SEQ. ID. No. 68:5'-GTGAACAGCAGTTGAACATGG
and SEQ. ID. No. 69:5'-CTTCACAAAGAAAAGAGAACTCTCCC), 5.8S (SEQ. ID.
No. 70: 5'-CGACTCTTAGCGGTGGATCA and SEQ. ID. No. 71:
5'-AAGCGACGCTCAGACAG and 5S (SEQ. ID. NO. 5 and SEQ. ID. No. 72:
5'-AAAGCCTACAGCACCCGGTATTC) rRNA sequences.
The QPCR results are shown in Table 2 below:
TABLE-US-00004 TABLE 2 Percentage Ribosomal RNA Reduction Exemplary
Method RiboMinus .TM. Method Intact Hela Fragmented Intact Hela
Fragmented total RNA Hela total RNA total RNA Hela total RNA QPCR
Primer Sets (2.5 .mu.g) (2.5 .mu.g) (2.5 .mu.g) (2.5 .mu.g)
18S.5'(nt 100-nt 247) >99.9% >99.9% 93.30% 50% 18S.3' (nt
1544-nt 1663) >99.9% >99.9% 97.10% 81.10% 18S.F3/R3 (nt
1288-nt 1417) >99.9% >99.9% 96.40% 34% 18S.F4/R4 (nt 1818-nt
1937) >99.9% >99.9% 97.40% 88.30% 28S 3.5K (nt 1748-nt 1867)
>99.9% >99.9% 85.60% 0% 28S #2 (nt 1324-nt 1530) >99.9%
99.60% 92.80% 90.50% 28S.F5/R5 (nt 4341-nt 4456) >99.9%
>99.9% 93.80% 78.20% 28S.5'#2 (nt 2740-nt 2843) >99.9%
>99.9% 92.80% 29.30% 28S.F3/R3 (nt 2401-nt 2630) >99.9%
>99.9% 83.50% 0% 28S.F4/R4 (nt 3732-nt 3851) >99.9% >99.9%
82.30% 0% 5.8S (nt 1-nt 157) >99.9% >99.9% 99.40% 99.60% 5S
(nt 1-nt 121) 96.80% 97.80% 91.80% 88.90%
Clearly, from the results in Table 2, the exemplary method was
significantly more efficient at removal of all ribosomal RNA
sequences independent of the primer sets used for both 28S and 18S
rRNA sequences with either intact or fragmented total RNA. Whereas,
for the RiboMinus.TM. method with the fragmented RNA, there was
little or no ribosomal subtraction depending on the location of the
different primer sets for both 28S and 18S rRNA sequences.
Example 13
Significant Reduction of rRNA Background and Improvement in
Uniquely Mappable Reads Using rRNA Removal Method Disclosed Herein
Compared to the RiboMinus.TM. Method
[0197] Intact and partially fragmented Universal Human Reference
RNA (UHRR) (2.times.2.5 .mu.g each) were treated with either the
method as described in Example 9 above or the RiboMinus.TM.
Eukaryote Kit for RNA-Seq rRNA removal kit. The respective
rRNA-depleted samples were pooled and, for each, Illumina RNA-Seq
libraries were prepared in triplicate using rRNA-depleted RNA from
the equivalent of 1 .mu.g total RNA. Replicates of the respective
RNA-Seq libraries were pooled and sequencing was performed using
Illumina.RTM. GAIIx next generation sequencer with 36-nt reads. The
data were analyzed using Illumina's Pipeline Eland_rna Module and
CASAVA Software as well as the TopHat Software for mapping splice
junctions (see http://followed by
"tophat.cbcb.umd.edu/index.html"). The mapping results showed that
rRNA background was significantly reduced by the methods described
in Example 9 compared to the RiboMinus.TM. method (see Table 3
below). In addition, the uniquely mappable sequences not including
rRNA sequences were significantly increased (Table 3) in the sample
treated with the methods as described in Example 9 compared to the
RiboMinus.TM.. Furthermore, for the fragmented samples, the Example
9 methods considerably outperformed the competitive kit, both in
terms of reducing rRNA background and improving the uniquely
mappable sequences (Table 3).
TABLE-US-00005 TABLE 3 % Uniquely rRNA Removal % rRNA Mappable
Total RNA Sample Method Background Sequences Intact UHRR Example 9
1.4% 58.1% Intact UHRR RiboMInus .TM. 18.4% 51.4% Fragmented UHRR
Example 9 2.1% 59.6% Fragmented UHRR RiboMInus .TM. 63.3% 24.6%
Table 3 show that the rRNA removal methods described in Example 9
significantly reduces rRNA background and improves RNA-Seq results.
Intact and partially fragmented Universal Human Reference RNA
(UHRR) was treated with either the method from Example 9 or the
commercial kit (RiboMinus.TM. method). The rRNA-depleted RNAs were
then used to prepare RNA-Seq libraries that were sequenced on an
Illumina.RTM. GAIIx sequencer.
Example 14
Prophetic Example for Plant rRNA Removal
[0198] Plants generally comprises rRNA sequences corresponding to
chloroplast, mitochondrial and of nuclear origins. For chloroplast
origin, the rRNA comprise 23S, 16S, 5S and 4.5S sequences (e.g.
Arabidopsis thaliana; Accession # AP000423.1); for mitochondrial
origin, the rRNA comprise 18S and 5S sequences (e.g., Arabidopsis
thaliana; Accession # Y08501.2) and for nuclear origin, the rRNA
comprise 25/26S, 17/18S and 5.8S sequences (e.g. Arabidopsis
thaliana; AC006837.16). PCR templates corresponding to each of the
plant rRNA sequence could be synthesized (in full or in part), as
well as the respective biotinylated rRNA sequences as described in
Example 1 for E. coli.
[0199] The respective biotinylated antisense could then be mixed
either in a single ratio or several different ratios in order to
efficiently remove all rRNA sequences from the different plant
tissues (e.g. leaf, root, stem etc.) where it is known that the
representation of the different rRNA are present in varying
amounts, especially dependent on the chloroplast content. It is
contemplated that the various plant rRNA sequences will be
effectively removed similar to that described for human and E. coli
rRNA sequences as in Examples 5-12 herein.
[0200] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference
(including, but not limited to, journal articles, U.S. and non-U.S.
patents, patent application publications, international patent
application publications, gene bank accession numbers, internet web
sites, and the like) cited in the present application is
incorporated herein by reference in its entirety.
Sequence CWU 1
1
72120DNAArtificial SequenceSynthetic 1cctacctacc tggttgatcc
20251DNAArtificial SequenceSynthetic 2aattctaata cgactcacta
tagggagaga tccttccgca ggttcaccta c 51323DNAArtificial
SequenceSynthetic 3cgactcttag cggtggatca ctc 23451DNAArtificial
SequenceSynthetic 4aattctaata cgactcacta tagggagaga tccttccgca
ggttcaccta c 51523DNAArtificial SequenceSynthetic 5gtctacggcc
ataccaccct gaa 23650DNAArtificial SequenceSynthetic 6aattctaata
cgactcacta tagggagaaa gcctacagca cccggtattc 50724DNAArtificial
SequenceSynthetic 7aagcgactaa gcgtacacgg tgga 24852DNAArtificial
SequenceSynthetic 8aattctaata cgactcacta tagggagatt cctggaagca
gggcatttgt tg 52924DNAArtificial SequenceSynthetic 9caacaaatgc
cctgcttcca ggaa 241053DNAArtificial SequenceSynthetic 10aattctaata
cgactcacta tagggagaca cggttcatta gtaccggtta gct 531120DNAArtificial
SequenceSynthetic 11agagtttgat cctggctcag 201250DNAArtificial
SequenceSynthetic 12aattctaata cgactcacta tagggagagg aggtgatcca
accgcaggtt 501322DNAArtificial SequenceSynthetic 13tgcctggcgg
cagtagcgcg gt 221450DNAArtificial SequenceSynthetic 14aattctaata
cgactcacta tagggagatg cctggcagtt ccctactctc 501522DNAArtificial
SequenceSynthetic 15gacgtgctaa tctgcgataa gc 221627DNAArtificial
SequenceSynthetic 16atggattcag ttaatgatag tgtgtcg
271727DNAArtificial SequenceSynthetic 17ctgaaagcat ctaagcacga
aacttgc 271824DNAArtificial SequenceSynthetic 18cctatcaacg
tcgtcgtctt caac 241922DNAArtificial SequenceSynthetic 19gcctaacaca
tgcaagtcga ac 222024DNAArtificial SequenceSynthetic 20agctaccgtt
tccagtagtt atcc 242123DNAArtificial SequenceSynthetic 21cggaatcgct
agtaatcgtg gat 232224DNAArtificial SequenceSynthetic 22tcccgaaggt
taagctacct actt 242322DNAArtificial SequenceSynthetic 23tgcctggcag
ttccctactc tc 222424DNAArtificial SequenceSynthetic 24accaggttaa
cccgtatgtt ggct 242524DNAArtificial SequenceSynthetic 25accgatgttg
ttggtccact ggta 242620DNAArtificial SequenceSynthetic 26ctcagtaacg
gcgagtgaac 202720DNAArtificial SequenceSynthetic 27gcctcgatca
gaaggacttg 202820DNAArtificial SequenceSynthetic 28taccacaggg
ataactggct 202920DNAArtificial SequenceSynthetic 29taggaagagc
cgacatcgaa 203020DNAArtificial SequenceSynthetic 30cctacctggt
tgatcctgcc 203120DNAArtificial SequenceSynthetic 31ccaagtagga
gaggagcgag 203220DNAArtificial SequenceSynthetic 32cccagtaagt
gcgggtcata 203325DNAArtificial SequenceSynthetic 33tcactaaacc
atccaatcgg tagta 253423DNAArtificial SequenceSynthetic 34gatccttccg
caggttcacc tac 233522DNAArtificial SequenceSynthetic 35aagcctacag
cacccggtat tc 223624DNAArtificial SequenceSynthetic 36tcgacagtca
gccgcatctt cttt 243724DNAArtificial SequenceSynthetic 37accaaatccg
ttgactccga cctt 243825DNAArtificial SequenceSynthetic 38cacaagagga
agagagagac cctca 253924DNAArtificial SequenceSynthetic 39ttgatggtac
atgacaaggt gcgg 244020DNAArtificial SequenceSynthetic 40gaatcaccga
cctctctccc 204120DNAArtificial SequenceSynthetic 41cgactctcat
aacgacccgc 204220DNAArtificial SequenceSynthetic 42ccagaggtga
ccactttcaa 204321DNAArtificial SequenceSynthetic 43atgtggaaca
gagccttcct c 214420DNAArtificial SequenceSynthetic 44cacgttttct
cagctgcttg 204520DNAArtificial SequenceSynthetic 45ttcacctttt
catccaaggc 204620DNAArtificial SequenceSynthetic 46gtgtggtggt
gtgtgcctat 204720DNAArtificial SequenceSynthetic 47gagacatggt
cttgctccgt 204820DNAArtificial SequenceSynthetic 48tagctcagtg
gtagagcgca 204920DNAArtificial SequenceSynthetic 49gatttgctca
gcagcacgta 205020DNAArtificial SequenceSynthetic 50cacttgggga
cactttccag 205120DNAArtificial SequenceSynthetic 51tcagggaaaa
tgagccaatc 205219DNAArtificial SequenceSynthetic 52cttagaggga
caagtggcg 195320DNAArtificial SequenceSynthetic 53gtagggtagg
cacacgctga 205424DNAArtificial SequenceSynthetic 54gaaacttaaa
ggaattgacg gaag 245523DNAArtificial SequenceSynthetic 55gaatcgagaa
agagctatca atc 235621DNAArtificial SequenceSynthetic 56cgattggatg
gtttagtgag g 215727DNAArtificial SequenceSynthetic 57ccttgttacg
acttttactt cctctag 275820DNAArtificial SequenceSynthetic
58gccgaaacga tctcaaccta 205920DNAArtificial SequenceSynthetic
59cgccagttct gcttaccaaa 206019DNAArtificial SequenceSynthetic
60cggaccaagg agtctaaca 196123DNAArtificial SequenceSynthetic
61caggcatagt tcaccatctt tcg 236220DNAArtificial SequenceSynthetic
62ggagagggtg taaatctcgc 206320DNAArtificial SequenceSynthetic
63gccgacttcc cttacctaca 206422DNAArtificial SequenceSynthetic
64gtgtcagaaa agttaccaca gg 226525DNAArtificial SequenceSynthetic
65ggcgaattct gcttcacaat gatag 256626DNAArtificial SequenceSynthetic
66gggagtaact atgactctct taaggt 266721DNAArtificial
SequenceSynthetic 67ttggctgtgg tttcgctgga t 216821DNAArtificial
SequenceSynthetic 68gtgaacagca gttgaacatg g 216926DNAArtificial
SequenceSynthetic 69cttcacaaag aaaagagaac tctccc
267020DNAArtificial SequenceSynthetic 70cgactcttag cggtggatca
207117DNAArtificial SequenceSynthetic 71aagcgacgct cagacag
177223DNAArtificial SequenceSynthetic 72aaagcctaca gcacccggta ttc
23
* * * * *
References