U.S. patent application number 10/667126 was filed with the patent office on 2005-03-17 for methods and compositions for isolating small rna molecules.
This patent application is currently assigned to Ambion, Inc.. Invention is credited to Conrad, Richard C..
Application Number | 20050059024 10/667126 |
Document ID | / |
Family ID | 34118826 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059024 |
Kind Code |
A1 |
Conrad, Richard C. |
March 17, 2005 |
Methods and compositions for isolating small RNA molecules
Abstract
The present invention concerns the use of methods and
compositions for the isolation of small RNA molecules (100
nucleotides or fewer), such as microRNA and siRNA molecules. Such
molecules are routinely lost in commonly used isolation procedures
and therefore the present invention allows for a much higher level
of enrichment or isolation of these small RNA molecules.
Inventors: |
Conrad, Richard C.; (Austin,
TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Assignee: |
Ambion, Inc.
|
Family ID: |
34118826 |
Appl. No.: |
10/667126 |
Filed: |
September 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60490325 |
Jul 25, 2003 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
536/25.4 |
Current CPC
Class: |
C12N 15/1006 20130101;
C12N 15/1003 20130101; C07H 1/08 20130101 |
Class at
Publication: |
435/006 ;
536/025.4 |
International
Class: |
C12Q 001/68; C07H
021/02 |
Claims
What is claimed is:
1. A method for isolating small RNA molecules from cells
comprising: a) lysing the cells with a lysing solution to produce a
lysate; b) adding an alcohol solution to the lysate; c) applying
the lysate to a solid support; d) eluting small RNA molecules from
the solid support; and, e) using or characterizing the small RNA
molecules.
2. The method of claim 1, wherein the small RNA molecules include
miRNA, siRNA, snRNA, snoRNA, and/or tRNA molecules.
3. The method of claim 2, wherein the small RNA molecules are miRNA
molecules.
4. The method of claim 1, wherein at least 20% of the small RNA
molecules from the cells are isolated.
5. The method of claim 4, wherein at least 50% of the small RNA
molecules from the cells are isolated.
6. The method of claim 1, wherein the lysing solution comprises a
chaotropic agent or detergent.
7. The method of claim 6, wherein the lysing solution comprises a
chaotropic agent.
8. The method of claim 7, wherein the concentration of the
chaotropic agent in the lysing solution is at least about 2.0
M.
9. The method of claim 7, wherein the lysing solution comprises
guanidinium.
10. The method of claim 9, wherein the concentration of guanidinium
is at least about 2.0 M.
11. The method of claim 10, wherein the lysing solution further
comprises a detergent and a buffer.
12. The method of claim 11, wherein the concentration of the
detergent is about 0.1% to about 2%.
13. The method of claim 12, wherein the detergent is N-lauroyl
sarcosine.
14. The method of claim 11, wherein the concentration of the buffer
is about 10 mM to about 300 mM.
15. The method of claim 1, further comprising extracting small RNA
molecules from the lysate with an extraction solution comprising an
organic solvent prior to applying the lysate to the solid
support.
16. The method of claim 15, wherein the extraction solution
comprises phenol.
17. The method of claim 16, wherein the extraction solution further
comprises chloroform.
18. The method of claim 1, wherein the amount of alcohol solution
added to the lysate makes the lysate about 20% to about 70%
alcohol.
19. The method of claim 18, wherein the amount of alcohol solution
added to the lysate makes the lysate about 50% to 60% alcohol.
20. The method of claim 18, wherein the alcohol solution is added
to the lysate before extraction with an organic solvent.
21. The method of claim 1, further comprising washing the solid
support with a first wash solution after applying the lysate to the
solid support.
22. The method of claim 21, wherein the first wash solution
comprises a chaotropic agent.
23. The method of claim 22, wherein the chaotropic agent is
guanidinium and the first wash solution further comprises
alcohol.
24. The method of claim 21, further comprising washing the solid
support with a second wash solution after washing with the first
wash solution.
25. The method of claim 24, wherein the second wash solution
comprises alcohol.
26. The method of claim 1, wherein the small RNA molecules are
eluted from the solid support at a temperature of about 60.degree.
C. to about 100.degree. C.
27. The method of claim 1, wherein the small RNA molecules are
eluted from the solid support with a low-ionic-strength
solution.
28. The method of claim 27, wherein the ionic solution comprises up
to 10 mM salt.
29. The method of claim 1, wherein the solid support is a mineral
support or polymer support.
30. The method of claim 29, wherein the mineral support or polymer
support is a column comprising silica.
31. The method of claim 29, wherein the mineral or polymer support
is a set of beads made of an absorptive polymer.
32. The method of claim 31, wherein the set of beads are collected
by centrifugation, filtration, or magnetic capture.
33. The method of claim 30, wherein the silica is glass fiber.
34. The method of claim 1, further comprising passing the lysate
through the column by centrifugation or gas pressure.
35. The method of claim 1, further comprising capturing the eluted
small RNA molecules.
36. The method of claim 33, wherein the eluted small RNA molecules
are captured on a filter and then collected.
37. The method of claim 1, wherein the small RNA molecules are
single stranded.
38. The method of claim 1, wherein the small RNA molecules are
double stranded.
39. The method of claim 1, wherein the small RNA molecules have at
most 100 nucleotides or fewer.
40. The method of claim 39, wherein the small RNA molecules have at
most 70 nucleotides or fewer.
41. The method of claim 40, wherein the small RNA molecules have at
most 30 nucleotides or fewer.
42. A method for isolating miRNA or siRNA from a sample comprising:
a) obtaining a sample having miRNA or siRNA; b) adding an alcohol
solution to the sample; c) adding an extraction solution to the
sample; c) applying the sample to a mineral or polymer support; and
d) eluting the siRNA or miRNA from the mineral or polymer support
with an ionic solution.
43. The method of claim 42, wherein the sample is a cell
lysate.
44. The method of claim 43, wherein the cell lysate is produced by
adding a lysing solution comprising a chaotropic agent or detergent
to cells having miRNA or siRNA.
45. The method of claim 42, wherein the eluted sample is enriched
at least about 10-fold by mass for miRNA or siRNA.
46. A method for isolating miRNA molecules from a sample
comprising: a) adding an alcohol solution to the sample; b)
applying the sample to a mineral or polymer support; c) eluting
miRNA molecules from the support with an ionic solution; and d)
using or characterizing the miRNA molecules.
47. The method of claim 46, wherein the sample is a cell
lysate.
48. A method for isolating small RNA molecules from a sample
comprising: a) lysing cells in the sample with a lysing solution
comprising guanidinium, wherein a lysate with a concentration of at
least about 1 M guanidinium is produced; b) extracting small RNA
molecules from the lysate with an extraction solution comprising
phenol; c) adding to the lysate an alcohol solution for form a
lysate/alcohol mixture, wherein the concentration of alcohol in the
mixture is between about 35% to about 70%; d) applying the
lysate/alcohol mixture to a mineral or polymer support; e) eluting
the small RNA molecules from the mineral or polymer support with an
ionic solution; f) capturing the small RNA molecules; and g) using
the isolated small RNA molecules.
49. A kit for isolating small RNA molecules comprising: a) acid
phenol-chloroform; b) lysis/binding buffer; c) homogenate additive;
d) wash solution; e) elution solution; and f) filter cartridges
50. A method for isolating small RNA molecules from a sample
comprising: a) lysing cells in a lysing solution to produce a
lysate; b) extracting small RNA molecules from the lysate with an
extraction solution comprising phenol; c) adding to the lysate an
alcohol solution to form a lysate/alcohol mixture; d) applying the
lysate/alcohol mixture to a first solid support; e) collecting
flow-through lysate/alcohol mixture; f) adding to the flow-through
lysate/alcohol mixture an alcohol solution; g) applying the
lysate/alcohol mixture to a second solid support; and h) eluting
small RNA molecules from the solid support with an ionic
solution.
51. The method of claim 50, wherein the lysate/alcohol mixture
applied to the first solid support is between about 20% to about
35% alcohol.
52. The method of claim 50, wherein the lysate/alcohol mixture
applied to the second solid support is between about 35% to about
70% alcohol.
53. The method of claim 50, further comprising using or
characterizing the small RNA molecules.
Description
[0001] The present application claims priority to U.S. Provisional
Application Ser. No. 60/490,325, filed on Jul. 25, 2003, which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
molecular biology and biotechnology. More particularly, it concerns
methods and compositions for isolating small RNA molecules that are
typically 100 nucleotides or fewer, such as siRNA and miRNA, as
opposed to larger RNA or DNA molecules. The isolated small RNA
molecules can be used in subsequent studies or assays.
[0004] 2. Description of Related Art
[0005] The study of small RNAs-RNA molecules on the order of 100
nucleotides or fewer-from various tissues in many organisms, as
well as cultured cells, is an area of extreme interest now, and
promises to remain one for the future. These small RNAs include
microRNA molecules (miRNA) and small interfering RNA molecules
(siRNA), both of which can have a powerful effect on the expression
of a gene by virtue of hybridization to their target mRNA.
Additionally, these procedures would be applicable to isolating
small nuclear and small nucleolar RNAs (snRNAs and snoRNAs),
involved in mRNA and rRNA processing. The procedures could also be
used to isolate tRNAs along with 5S and 5.8S rRNAs, which are all
involved in protein translation.
[0006] Key to these studies is the need to isolate RNA molecules in
the size range of 15 to 100 nucleotides with high efficiency.
Methods that provide a straightforward methodology to do this are
therefore quite valuable.
[0007] The preparation of RNA from natural sources (tissue samples,
whole organisms, cell cultures, bodily fluids) requires removal of
all other biomolecules. Once water is eliminated, the primary
component of cells is usually protein, often providing
three-quarters of the mass. Of the major other biomolecules,
lipids, carbohydrates, combinations of these with each other and
protein, and DNA are the other main components. A goal of RNA
extraction is to remove protein and DNA, as these provide the
greatest interference in the use of RNA. Lipid and carbohydrate
moieties can usually be dissolved away with the aid of a detergent.
Protein can be stripped off RNA (and DNA) with the aid of
detergents and denaturants, but still must be removed from the
common solution.
[0008] Two main methods have historically been used to accomplish
this end. The first is the use of organic solvents that are
immiscible with water to dissolve (literally, to chemically
extract) or precipitate proteins, after which the aqueous,
protein-free phase can be separated by centrifugation prior to
removal. Usually, phenol or phenol-chloroform mixtures are used for
this purpose. The second method selectively immobilizes the RNA on
a solid surface and rinses the protein away, after which conditions
are used to release the RNA in an aqueous solution. This is
literally a solid-phase extraction. Both procedures can reduce the
amount of DNA contamination or carryover, with the efficiency
varying with the precise conditions employed.
[0009] Phenol and phenol-chloroform extractions provide an
extremely protein- and lipid-free solution of nucleic acid. Much if
not all (depending on the sample) of the carbohydrate is also lost
in this procedure as well. Acid phenol-chloroform is known to
extract some of the DNA out of the aqueous solution (Chomczynski
and Sacchi, 1987). However, the solution is high in denaturing
agents such as guanidinium hydrochloride, guanidinium thiocyanate,
or urea, all of which are incompatible with downstream enzymatic
analysis, and the first two with electrophoretic analysis as well.
RNA is usually separated from these mixtures by selective
precipitation, usually with ethanol or isopropanol. This procedure
is not as effective for small nucleic acid molecules, so this
procedure is not ideal for the preparation of small RNAs.
[0010] Solid-phase extraction relies on high salt or salt and
alcohol to decrease the affinity of RNA for water and increase it
for the solid support used. The use of glass (silica) as a solid
support has been shown to work for large RNAs in the presence of
high concentrations of denaturing salts (U.S. Pat. Nos. 5,155,018;
5,990,302; 6,043,354; 6,110,363; 5,234,809; Boom et al., 1990) or
lower concentrations of denaturing salts plus ethanol (U.S. Pat.
No. 6,180,778). However, normal conditions for binding to glass
fiber or RNA do not work for microRNA, and the use of a raw lysate
is problematic due to variable requirements with different
tissues.
[0011] Many of the protocols known involve isolation of DNA or
larger mRNA, which are not ideal for isolation of small RNA
molecules because these are often not effectively captured and
eluted. Thus, there is a need for improved techniques for the
efficient isolation, detection, and accurate quantification of
these recently discovered small RNA molecules.
SUMMARY OF THE INVENTION
[0012] The present invention concerns methods and compositions for
isolating, extracting, purifying, characterizing, quantifying,
and/or assaying small RNA molecules from a sample, including a cell
sample. Such compositions and methods allow for manipulation of
small RNA molecules, which are often lost or depleted when methods
for generally isolating larger RNA molecules are employed.
[0013] Thus, it is contemplated that the invention concerns small
RNA molecules, which are, in most embodiments, understood to be RNA
molecules of about 100 nucleotides or fewer. Small RNA molecules
include siRNA and miRNA molecules. In some embodiments of the
invention, the small RNA molecules have at most 100 nucleotides or
fewer, have at most 70 nucleotides or fewer, or have at most 30
nucleotides or fewer, or have at most 25, 24, 23, 22, 21, 20, 19,
18, 17, 16, 15 nucleotides or fewer.
[0014] In some cases, the small RNA molecules are double stranded.
In some cases, the small RNA molecules are single stranded, though
they may have regions of self-complementary. There may be 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more such regions, and these regions may
involve 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more
basepairs (and thus, twice as many bases).
[0015] Furthermore, these regions of complementary may involve 100%
complementary or it may involve some mismatches, such as at least
70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity in the
region among bases, or a region may contain 1, 2, 3, 4, 5, 6, 7, 8,
9 10 or more mismatches among bases in the region.
[0016] It is specifically contemplated that methods and
compositions of the invention can be used to isolate small RNA
molecules that are at most 100, 95, 90, 85, 80, 75, 70, 65, 60, 55,
50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 10
or fewer nucleotides in length, and all ranges derivable between
these integers. Furthermore, such molecules can be isolated so that
a sample is enriched in the amount of small RNA molecules
present.
[0017] There are several ways in which enrichment and/or
purification of small RNAs may be expressed in the context of the
invention. Any increase in the amount of small RNA molecules
present in a sample is within the scope of the invention.
[0018] Enrichment and/or purification of small RNAs may be measured
in terms of mass of small RNA relative to mass of total RNA. For
example, small RNA in a sample may be enriched about or at least
about 1.times., 1.5.times., 2.times., 2.5.times., 3.times.,
3.25.times., 3.5.times., 3.75.times., 4.times., 4.25.times.,
4.5.times., 4.75.times., 5.times., 5.25.times., 5.5.times.,
5.75.times., 6.times., 6.25.times., 6.5.times., 6.75.times.,
7.times., 7.25.times., 7.5.times., 7.75.times., 8.times.,
8.25.times., 8.5.times., 8.75.times., 9.times., 9.5.times.,
10.times., 15.times., 20.times., 25.times., 30.times., 35.times.,
40.times., 45.times.. 50.times.. 55.times., 60.times., 65.times.,
70.times., 75.times., 80.times., 85.times., 90.times., 95.times.,
100.times., 110.times., 120.times., 130.times., 140.times.,
150.times., 160.times., 170.times., 180.times., 190.times.,
200.times., 210.times., 220.times., 230.times., 240.times.,
250.times., 260.times., 270.times., 280.times., 290.times.,
300.times., 325.times., 350.times., 375.times., 400.times.,
425.times., 450.times., 475.times., 500.times., 525.times.,
550.times., 575.times., 600.times., 625.times., 650.times.,
675.times., 700.times., 725.times., 750.times., 775.times.,
800.times., 825.times., 850.times., 875.times., 900.times.,
925.times., 950.times., 975.times., 1000.times., 1100.times.,
1200.times., 1300.times., 1400.times., 1500.times., 1600.times.,
1700.times., 1800.times., 1900.times., 2000.times. (same as -fold),
and all ranges derivable therein in small RNA molecules as
determined by the mass of small RNA molecules relative to the mass
of total RNA molecules prior to placing the lysate on the solid
support compared to after eluting the RNA from the solid
support.
[0019] Enrichment and/or may, alternatively, be measured in terms
of the number of small RNA molecules relative to the number of
total RNA molecules. Small RNA molecules can be isolated such that
a sample is enriched about or at least about 2.times., 3.times.,
4.times., 5.times., 10.times., 15.times., 20.times., 25.times.,
30.times., 35.times., 40.times., 45.times.. 50.times.. 55.times.,
60.times., 65.times., 70.times., 75.times., 80.times., 85.times.,
90.times., 95.times., 100.times., 1100.times., 120.times.,
130.times., 140.times., 150.times., 160.times., 170.times.,
180.times., 190.times., 200.times., 210.times., 220.times.,
230.times., 240.times., 250.times., 260.times., 270.times.,
280.times., 290.times., 300.times., 325.times., 350.times.,
375.times., 400.times., 425.times., 450.times., 475.times.,
500.times., 525.times., 550.times., 575.times., 600.times.,
625.times., 650.times., 675.times., 700.times., 725.times.,
750.times., 775.times., 30 800.times., 825.times., 850.times.,
875.times., 900.times., 925.times., 950.times., 975.times.,
1000.times., 1100.times., 1200.times., 1300.times., 1400.times.,
500.times., 1600.times., 1700.times., 1800.times., 1900.times.,
2000.times. (same as -fold) and all ranges derivable herein in
small RNA molecules as determined by number of small RNA molecules
relative to total number of RNA molecules prior to placing the
lysate on the solid support compared to after eluting the RNA from
the solid support.
[0020] Enrichment and/or purification of small RNAs may also be
measured in terms of the increase of small RNA molecules relative
to the number of total RNA molecules. Small RNA molecules can be
isolated such that the amount of small RNA molecules is increased
about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more with
respect to the total amount of RNA in the sample before and after
isolation.
[0021] Alternatively, in some embodiments, the enrichment and/or
purification of small RNA molecules can be quantified in terms of
the absence of large RNA molecules present in the sample after
eluting the RNA from the solid support. Small RNA molecules can be
enriched such that the number of RNA molecules larger than 200
nucleotides by mass remaining in the sample after eluting the RNA
from the solid support is no more than about 30%, 25%, 20%, 15%,
10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0%, or any range therein of the RNA
eluted from the solid support.
[0022] In some embodiments, about or at least about 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% of the small RNA molecules in a sample is isolated after
the method is implemented.
[0023] Methods of the invention include methods for efficiently
isolating small RNA molecules from cells comprising: a) lysing the
cells with a lysing solution to produce a lysate; b) adding an
alcohol solution to the lysate; c) applying the lysate to a solid
support; and d) eluting RNA molecules from the solid support.
[0024] Because the small RNA molecules are being efficiently
isolated, methods of the invention include a step of e) using or
characterizing the small RNA molecules. Using or characterizing the
small RNA molecules is distinguished from discarding the small RNA
molecules or having them as a byproduct or contaminant in a
reaction or assay involving other types of molecules isolated from
the sample, such as longer RNA molecules or DNA molecules.
[0025] Samples from which small RNA molecules may be isolated
include any sample containing such molecules. The sample may be or
contain cells, tissue, organs, or other biological sample.
Alternatively, the sample may be a reaction mixture, such as one in
which small RNA molecules were produced, generated, or created by
enzymatic, synthetic, and/or recombinant means.
[0026] In some embodiments, methods of the invention involve lysing
a sample that contains cells. A "lysate" results when a cell is
lysed or its integrity disrupted. In specific embodiments of the
invention, a lysing solution is implemented to lyse a cell sample,
and the solution includes a chaotropic agent or detergent. A
"chaotropic agent" refers to an agent that unfolds ordered
macromolecules, thereby causing them to lose their function (hence
causing binding proteins to release their target). A "detergent"
refers to a substance that can disperse a hydrophobic substance
(usually lipids) in water by emulsification. The concentration of a
chaotropic agent in the solutions of the invention, particularly
lysing solutions, is about, is at most about, or is at least about
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,
3.1, 3.2, 3.3, 3.4, 3.5 M or more, and ranges therein. In specific
embodiments, the concentration of guanidinium in the lysing
solution is between about 2.0 M and 4.0 M. In some embodiments, the
chaotropic agent is guanidinium chloride or guanidinium
isothiocyanate. In still further embodiments, the lysing solution
also contains a detergent and/or buffer. The concentration of the
detergent is between 0.1% to about 2% in some embodiments. The
detergent, particularly a mild one that is nondenaturing, can act
to solubilize the sample. Detergents may be ionic or nonionic.
[0027] The ionic detergent N-lauroyl sarcosine is specifically
contemplated for use in solutions of the invention. The
concentration of the detergent in the buffer may be about, at least
about, or at most about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%,
0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%,
2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%,
8.0%, 8.5%, 9.0%, 9.5%, 10.0% or any range therein. It is
contemplated that the concentration of the detergent can be up to
an amount where the detergent remains soluble in the solution.
[0028] In other embodiments of the invention, there is a buffer in
solutions of the invention, including a lysing solution. In
specific embodiments, the buffer is at a concentration of about, at
least about, or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,
125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,
190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,
255, 260, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340,
350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,
480, 490, 500 mM or any range therein in the solution or in the
solution with the sample. In certain cases, the buffer
concentration in the lysing solution is between about 10 mM and 300
mM. Moreover, in other embodiments, the buffer is TrisCl, although
it is contemplated that other buffers may be employed as well.
[0029] An alcohol solution is added to, mixed with, or incubated
with the lysate in embodiments of the invention. An alcohol
solution is contemplated to contain at least one alcohol. The
alcohol solution can be about, be at least about, or be at most
about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, or 100% alcohol, or any range therein. In certain
embodiments, it is added to a lysate to make the lysate have a
concentration of alcohol of about, about at least, or about at most
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or
90%, or any range therein. In specific embodiments, the amount of
alcohol added to a lysate renders it with an alcohol concentration
of about 35% to about 70%, or about 50% to about 60%. In specific
embodiments, the amount of alcohol solution added to the lysate
gives it an alcohol concentration of 55%. Alcohols include, but are
not limited to, ethanol, propanol, isopropanol, and methanol.
Ethanol is specifically contemplated for use in aspects of the
invention. It is further contemplated that an alcohol solution may
be used in additional steps of methods of the invention to
precipitate RNA.
[0030] It is contemplated that the pH of any solution, or of the
buffer component of any solution, or of any solution with the
sample is between about 4.5 and 10.5, though it can be about, about
at least, or about at most 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2
,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,
6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,
7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3,
10.4, 10.5 or any range therein.
[0031] Other methods of the invention also include extracting small
RNA molecules from the lysate with an extraction solution
comprising a non-alcohol organic solvent prior to applying the
lysate to the solid support. In specific embodiments, the
extraction solution contains a non-alcohol organic solvent such as
phenol and/or chloroform. The non-alcohol organic solvent solution
is understood to contain at least one non-alcohol organic solvent,
though it may also contain an alcohol. The concentrations described
above with respect to alcohol solutions are applicable to
concentrations of solutions having non-alcohol organic solvents. In
specific embodiments, equal amounts of 1) the lysate and 2) phenol
and/or chloroform are mixed. In specific embodiments, the alcohol
solution is added to the lysate before extraction with a
non-alcohol organic solvent.
[0032] Extraction of RNA from the lysate includes using a solid
support, such as a mineral or polymer support. A "solid support"
refers to a physical structure containing a material which contacts
the lysate and that does not irreversibly react to macromolecules
in the lysate, particularly with small RNA molecules In particular
embodiments, the solid support binds small RNA molecules; in
additional cases, it binds small RNA molecules, but does not bind
one or more other types of macromolecules in the sample. The
material in the solid support may include a mineral or polymer, in
which case the support is referred to as a "mineral or polymer
support." Mineral or polymer supports include supports involving
silica. In some embodiments, the silica is glass. Supports include,
but are not limited to, beads, columns and filters. In further
embodiments, the mineral or polymer support is a glass fiber filter
or column.
[0033] Alternatively, in some embodiments, the mineral or polymer
support may include polymers or nonpolymers with electronegative
groups. In some embodiments, the material is or has polyacrylate,
polystyrene, latex, polyacrylonitrile, polyvinylchloride,
methacrylate, and/or methyl methacrylate. Such supports are
specifically contemplated for use with the present invention.
[0034] In some methods of the invention, a lysate that may or may
not have been mixed with an alcohol or non-alcohol organic solvent
solution is applied to a solid support and the RNA is eluted from
the support.
[0035] After a lysate is applied or mixed with a solid support, the
material may be washed with a solution. In some embodiments, a
mineral or polymer support is washed with a first wash solution
after applying the lysate to the mineral or polymer support. In
further embodiments, a wash solution comprises a chaotropic or
reducing agent. The chaotropic agent is guanidinium in some wash
solutions. A wash solution includes alcohol in some embodiments of
the invention, and in some cases, it has both alcohol and
guanidinium. It is further contemplated that methods of the
invention involve 1, 2, 3, 4, 5 or more washes with a wash
solution. The wash solution used when more than one washing is
involved may be the same or different. In some embodiments, the
wash solutions have the same components but in different
concentrations from each other. It is generally understood that
molecules that come through the material in a wash cycle are
discarded.
[0036] In other methods of the invention, the desired RNA molecules
are eluted from the solid support. In certain embodiments, small
RNA molecules are eluted from a solid support such as a mineral or
polymer support at a temperature of about 60.degree. C. to about
100.degree. C. It is contemplated that the temperature at which the
RNA molecules are eluted is about or at least about 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100.degree. C. or more, or any range therein. The molecules may be
eluted with any elution solution. In some embodiments, the elution
solution is an ionic solution, that is, it includes ions. In
particular embodiments, the elution solution includes up to 10 mM
salt. It is contemplated to be about, at least about, or at most
about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mM salt. In
certain embodiments, the salt consists of a combination of
Li.sup.+, Na.sup.+, K.sup.+, or NH.sub.4.sup.+ as cation and
Cl.sup.-, Br.sup.-, I.sup.-, ethylenediaminetetraacetate, or
citrate as anion.
[0037] Additional method steps include passing the small RNA
molecules through a GFF while binding only the larger RNAs. In some
embodiments, the passed small RNA molecules are captured on a
second GFF and then eluted. Material that is not captured on the
second GFF filter is discarded or not used in additional methods of
the invention.
[0038] Specific methods of the invention concern isolating miRNA or
siRNA from a sample by at least the following steps: a) obtaining a
sample having miRNA or siRNA; b)adding an extraction solution to
the sample; c) adding an alcohol solution to the extracted sample;
d) applying the sample to a mineral or polymer support; and, e)
eluting the RNA containing siRNA or miRNA from the mineral or
polymer support with an ionic solution. In particular embodiments,
the sample is a cell lysate. The cell lysate, in some cases, is
produced by adding a lysing solution comprising a chaotropic agent
or detergent to cells having miRNA or siRNA. In some embodiments,
the eluted sample is enriched at least about 10-fold for miRNA
and/or siRNA by mass.
[0039] Additional methods for isolating miRNA molecules from a
sample involve: a) adding an alcohol solution to the sample; b)
applying the sample to a mineral or polymer solid support; c)
eluting miRNA molecules from the support with an ionic solution;
and, d) using or characterizing the miRNA molecules.
[0040] Other methods for isolating small RNA molecules from a
sample include: a) lysing cells in the sample with a lysing
solution comprising guanidinium, wherein a lysate with a
concentration of at least about 1 M guanidinium is produced; b)
extracting small RNA molecules from the lysate with an extraction
solution comprising phenol; c) adding to the lysate an alcohol
solution for form a lysate/alcohol mixture, wherein the
concentration of alcohol in the mixture is between about 35% to
about 70%; d) applying the lysate/alcohol mixture to a solid
support; e) eluting the small RNA molecules from the solid support
with an ionic solution; f) capturing the small RNA molecules; and,
g) using the isolated small RNA molecules.
[0041] After RNA is extracted, individual or specific RNA molecules
and/or pools of RNA molecules (as well as the entire population of
isolated RNA) can be subject to additional reactions and/or assays.
In some cases, these reactions and/or assays involve amplification
of the RNA or of a DNA molecule generated from the RNA. For
example, RT-PCR may be employed to generate molecules that can be
characterized.
[0042] In some embodiments, a particular RNA molecule or an RNA
population may be quantified or characterized. Quantification
includes any procedure known to those of skill in the art such as
those involving one or more amplification reactions or nuclease
protection assays, such as those using ribonuclease to discriminate
between probe that is hybridized to a specific miRNA target or
unhybridized, as embodied in the mirVana miRNA Detection Kit from
Ambion. These procedures include quantitative reverse
transcriptase-PCR (qRT-PCR). In some embodiments, characterization
of the isolated RNA is performed. cDNA molecules are generated from
the extracted RNA. Other characterization and quantification assays
are contemplated as part of the invention. The methods and
compositions of the invention allow small RNA molecules to be
quantified and characterized. The small RNA molecules can also be
used with arrays; to generate cDNAs for use in arrays or as targets
to be detected by arrays, after being labeled by radioactive,
fluorescent, or luminescent tags. Other assays include the use of
spectrophotometry, electrophoresis, and sequencing.
[0043] The present invention also concerns kits for isolating small
RNA molecules, such as miRNA and/or siRNA from a sample,
particularly a cell sample. Thus, any of the compositions discussed
above can be included with any other composition discussed above
for inclusion in a kit. In some embodiments, there are kits for
isolating small RNA molecules comprising: a) acid
phenol-chloroform; b) a lysis/binding buffer, c) a small RNA
homogenate additive, d) one or more small RNA wash solution(s), and
e) an elution solution.
[0044] In preferred embodiment, the kit contains: a) an acid
phenol-chloroform; b) a lysis/binding buffer comprising 4 M GuSCN,
0.1 M beta-mercaptoethanol, 0.5% N-lauroyl sarcosine, 25 mM
Na-citrate, pH 7.2; c) a small RNA homogenate additive comprising 2
M sodium acetate, pH 4, to be added in 0.1 volume before extraction
with PC; d) a wash solution #1 comprising 1.6 M GuSCN in 70%
ethanol; e) a wash solution #2/3 comprising 80% ethanol, 0.1 M
NaCl, 4.5 mM EDTA, 10 mM TrisHCl, pH 7.5; f) an elution solution
comprising 0.1 mM EDTA, pH8; g) a gel loading buffer II; h)
collection tubes; and i) filter cartridges.
[0045] In some embodiments, kits of the invention include one or
more of the following n a suitable container means (consistent with
compositions discussed above): a lysis buffer with a chaotropic
agent; a glass fiber filter or column; elution buffer; wash buffer;
alcohol solution; and RNase inhibitor.
[0046] The components of the kits may be packaged either in aqueous
media or in lyophilized form. The container means of the kits will
generally include at least one vial, test tube, flask, bottle,
syringe or other container means, into which a component may be
placed, and preferably, suitably aliquoted. Where there are more
than one component in the kit (they may be packaged together), the
kit also will generally contain a second, third or other additional
container into which the additional components may be separately
placed. However, various combinations of components may be
comprised in a vial. The kits of the present invention also will
typically include a means for containing the RNA, and any other
reagent containers in close confinement for commercial sale. Such
containers may include injection or blow-molded plastic containers
into which the desired vials are retained. When the components of
the kit are provided in one and/or more liquid solutions, the
liquid solution is an aqueous solution, with a sterile aqueous
solution being particularly preferred.
[0047] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means. The container means will
generally include at least one vial, test tube, flask, bottle,
syringe and/or other container means, into which the nucleic acid
formulations are placed, preferably, suitably allocated. The kits
may also comprise a second container means for containing a
sterile, pharmaceutically acceptable buffer and/or other
diluent.
[0048] Such kits may also include components that preserve or
maintain the RNA or that protect against its degradation. Such
components may be RNAse-free or protect against RNAses. Such kits
generally will comprise, in suitable means, distinct containers for
each individual reagent or solution.
[0049] It is contemplated that any embodiment discussed herein can
be implemented with respect to any method or composition of the
invention, and vice versa. Furthermore, compositions and kits of
the invention can be used to achieve methods of the invention.
[0050] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0051] Following long-standing patent law, the words "a" and "an,"
when used in conjunction with the word "comprising" in the claims
or specification, denotes one or more, unless specifically
noted.
[0052] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0054] FIG. 1. Binding behavior of let-7 miRNA in raw extracts from
mouse brain, heart, and liver at various ethanol concentrations.
Absolute ethanol was added to crude lysate to create the final
ethanol concentrations indicated.
[0055] FIG. 2. Binding behavior of let-7 miRNA in phenol-chloroform
extracts from mouse brain, heart, and liver at various ethanol
concentrations. Absolute ethanol was added to lysates after
extraction by phenol-chloroform (as in the standard procedure) to
create the final ethanol concentrations indicated.
[0056] FIG. 3. Binding behavior of .beta.-actin, GAPDH, PPI, U2,
and let-7 at varying ethanol concentrations in the presence of 2M
GuSCN, with ethanol concentration ajusted by addition of an equal
volume of a 2.times. ethanol solution in water.
[0057] FIG. 4. Binding behavior of .beta.-actin, GAPDH, PPI, U2,
and let-7 at varying ethanol concentrations in the presence of 2M
GuSCN, with ethanol concentration adjusted by addition of absolute
ethanol.
[0058] FIG. 5. Binding behavior of .beta.-actin, GAPDH, PPI, U2,
and let-7 at varying ethanol concentrations in the presence of 3M
GuSCN, with ethanol concentration adjusted by addition of an equal
volume of a 2.times. ethanol solution in water.
[0059] FIG. 6. Binding behavior of .beta.-actin, GAPDH, PPI, U2,
and let-7 at varying ethanol concentrations in the presence of 3M
GuSCN, with ethanol concentration adjusted by addition of absolute
ethanol.
[0060] FIG. 7. Relative enrichment of .beta.-actin, GAPDH, U2, and
let-7 RNAs.
[0061] FIG. 8. Relative enrichment of U2 and let 7 RNAs.
[0062] FIG. 9. Yield of current procedure compared to standard
phenol-chloroform extraction and ethanol precipitation.
[0063] FIG. 10. Comparison of absolute yield of three small RNAs,
let-7 (22nt), U43 (62nt), and U2 (187nt), using the current process
(Ambion microRNA Isolation Kit=AMIK) versus a glass fiber system
currently available (RNeasy).
[0064] FIG. 11. Comparison of yield from cultured cells, with and
without pre-extraction. The yield of both U2 and let-7 were
determined.
[0065] FIG. 12. Effects of different concentrations of NaOOCH.sub.3
at two pHs for the phenol-hloroform extraction prior to glass
immobilization on yield of U2, U43, and let-7.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. Small RNA Molecules
[0066] Natural populations of RNA are routinely isolated from
animal and plant tissue as well as cells grown in culture. However,
most of these procedures are unconcerned with retaining small RNAs,
in the range of less than 100 nucleotides long. In fact, it is
known that standard precipitation procedures with alcohol are
inefficient in capturing nucleic acids smaller than around 100
nucleotides.
[0067] The presence of small RNA molecules and free nucleotides has
long been observed in RNA extracted from biological samples and
assumed to reflect the breakdown products of larger protein-coding
and functional RNAs, including those involved in translation and
RNA processing complexes. In the past few years, small RNAs
involved in the regulation of gene expression have been found to be
present in virtually all eukaryotic organisms. In 1993, the Ambrose
lab published a report on the discovery that the let-7gene, which
results in developmental mis-timing, or heterochromy, in the
nematode Caenorhabditis elegans coded for a 22-nt RNA (Lee et al.,
1993.). This small, single-stranded RNA (now termed microRNA or
miRNA) affects the expression of a set of developmental genes by
inhibiting their ability to function in translation based on
partial sequence complementary with the targeted gene. The presence
of this small RNA was found to be conserved in many evolutionarily
divergent species (Pasquinelli et al., 2000), including vertebrate,
ascidian, hemichordate, mollusc, annelid and arthropod.
[0068] In 2001, several groups used a novel cloning method to
isolate and identify a large variety of these "micro RNAs" (miRNAs)
from C. elegans, Drosophila, and humans (Lagos-Quintana et al.,
2001; Lau et al., 2001; Lee and Ambros, 2001). Several hundreds of
miRNAs have been identified in plants and animals. miRNAs thus far
observed have been approximately 21-22 nucleotides in length and
they arise from longer precursors, which are transcribed from
non-protein-encoding genes. See review of Carrington et al. (2003).
The precursors form structures that fold back on each other in
self-complementary regions; they are then processed by the nuclease
Dicer in animals or DCL1 in plants. miRNA molecules interrupt
translation through imprecise base-pairing with their targets.
[0069] Micro RNAs are not the only RNAs of that size found in
eukaryotic cells. A pathway for degradation of mRNAs in the cell
was found that creates small double-stranded RNAs (Fire et al.,
1998; Zamore et al., 2000; many others, reviewed in Timmons,
2002.). This process, called RNA interference, uses these "small
interfering RNAs" (siRNAs) to target their degradation sites,
usually from a much larger double-stranded intermediate. Although
the natural function of this system is not known, it is thought to
be involved in the response to infective agents. Plants have been
found to have a similar system, which also utilizes microRNAs in
post-transcriptional gene-silencing (Hamilton and Baulcombe, 1999;
Tang et al. 2003).
II. Isolation of Short RNA Molecules
[0070] Methods of the invention involve one or more steps to
efficiently isolate and/or enrich short RNA molecules. These steps
include or involve the following: lysing cells and/or creating a
cell lysate;
[0071] A. Creating Cell Lysates
[0072] It is contemplated that the present invention can be used to
facilitate preparation of small RNA molecules from biological
samples for evaluation and subsequent use. In some embodiments of
the invention, preparation of samples involves homogenizing the
sample or preparing a cell lysate from the sample. In embodiments
of the invention, homogenization or lysing of a cell is
accomplished using a solution that contains a guanidinium salt,
detergent, surfactant, or other denaturant. The terms
homogenization and lysing are used interchangeably.
[0073] Guanidinium salts are well known to those of skill in the
art and include guanidinium hydrochloride and guanidinium
isothiocyanate. In some embodiments, they may be present in a
concentration of about 2 to about 5 M. Additionally, a
homogenization solution may contain urea or other denaturants such
as NaI.
[0074] In embodiments of the invention, a buffer is included in the
lysis or homogenization solution. In certain cases, the buffer is
TrisCl.
[0075] A biological sample may be homogenized or fractionated in
the presence of a detergent or surfactant. The detergent can act to
solublize the sample. Detergents may be ionic or nonionic. Examples
of nonionic detergents include triton, such as the Triton X series
(Triton X-100, Triton X-100R, Triton X-114, Triton X-450, Triton
X-450R), octyl glucoside, polyoxyethylene(9)dodecyl ether,
digitonin, IGEPAL CA630, n-octyl-beta-D-glucopyranoside (betaOG),
n-dodecyl-beta, C12EO7, Tween 20, Tween 80, polidocanol, n-dodecyl
beta-D-maltoside (DDM), NP-40, C12E8 (octaethylene glycol n-dodecyl
monoether), hexaethyleneglycol mono-n-tetradecyl ether (C14EO6),
octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG), Emulgen,
and polyoxyethylene 10 lauryl ether (C12E10). Examples of ionic
detergents (anionic or cationic) include deoxycholate, sodium
dodecyl sulfate (SDS), N-lauroylsarcosine, and
cetyltrimethylammoniumbromide (CTAB). A zwitterionic reagent may
also be used in the purification schemes of the present invention,
such as Chaps, zwitterion 3-14, and
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulf- onate. It is
contemplated also that urea may be added with or without another
detergent or surfactant.
[0076] Lysis or homogenization solutions may further contain other
agents such as reducing agents. Examples of such reducing agents
include dithiothreitol (DTT), .beta.-mercaptoethanol, DTE, GSH,
cysteine, cysteamine, tricarboxyethyl phosphine (TCEP), or salts of
sulfurous acid.
[0077] In some embodiments of the invention, a lysis solution
includes: guanidinium thiocyanate, N-lauroyl sarcosine, and
TrisHCl. Once the sample has been homogenized into this solution,
the RNA can be extracted, often with phenol solutions or the use of
an adsorptive solid phase. Alternative methods use combination
denaturant/phenol solutions to perform the initial homogenization,
precluding the need for a secondary extraction. Examples of these
reagents would be Trizol.TM. (Invitrogen) or RNAwiz.TM. (Ambion,
Inc.)
[0078] Subsequent to exposure to a homogenization solution, samples
may be further homogenized by mechanical means. Mechanical
blenders, rotor-stator homogenizers, or shear-type homogenizers may
be employed.
[0079] Alternatively, the tissue could be homogenized in the lysis
solution, and the tissue remains separated by settling,
centrifugation, or filtration. These remains could then be treated
with homogenization solution and extraction conditions as described
above.
[0080] The methods of the invention may further include steps
involving removing lipids or compositions thereof with detergents
or surfactants. A lipid may be naturally occurring or synthetic
(i.e., designed or produced by man). However, a lipid is usually a
biological substance. Biological lipids are well known in the art,
and include for example, neutral fats, phospholipids,
phosphoglycerides, steroids, terpenes, lysolipids,
glycosphingolipids, glucolipids, sulphatides, lipids with ether and
ester-linked fatty acids and polymerizable lipids, and combinations
thereof. Removal of a lipid such as a phospholipid is described
herein.
[0081] Detergents may be used to facilitate homogenization or the
creation of a cell lysate. These detergents specifically include
Triton X-100 and CHAPS. CHAPS is the zwitterionic detergent
3-[(3-cholamidopropyl)-dimethy- l-ammonio]-1-propanesulfonate.
[0082] B. Extracting Small RNA Molecules
[0083] After lysing or homogenizing a cell sample, additional
procedures may be implemented to extract specifically RNA
molecules. It is contemplated that if the sample involves cells,
the step of lysing or homogenizing can be considered part of an
overall extraction process, however, the extraction of RNA
specifically may be referred to, and will be understood as
separating RNA molecules from other biomolecules such as lipids and
proteins. Extraction of RNA molecules from these other structures
can involve extraction solutions containing one or more organic
solvents. In some cases, the organic solvent is a non-alcohol
organic solvent such as phenol and/or chloroform. In others, a
solution contains an alcohol, which may be any alcohol used for the
extraction of nucleic acids, but in certain embodiments, the
alcohol is ethanol.
[0084] RNA molecules may be extracted from a variety of cell
samples. Such cell samples may comprise cells of the brain, head,
neck, gastrointestinal tract, lung, liver, pancreas, breast,
testis, uterus, bladder, kidney, prostate, colon, kidney, skin,
ovary, and heart but is not limited to such cells.
[0085] The term "nucleic acid" is well known in the art. A "nucleic
acid" as used herein will generally refer to a molecule (i.e., a
strand) of DNA, RNA or a derivative or analog thereof, comprising a
nucleobase. A nucleobase includes, for example, a naturally
occurring purine or pyrimidine base found in DNA (e.g., an adenine
"A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g.,
an A, a G, an uracil "U" or a C). The term "nucleic acid" or "RNA
molecule" encompasses the terms "oligonucleotide" and
"polynucleotide," each as a subgenus of the term "nucleic
acid."
[0086] A nucleic acid "complement(s)" or is "complementary" to
another nucleic acid when it is capable of base-pairing with
another nucleic acid according to the standard Watson-Crick,
Hoogsteen or reverse Hoogsteen binding complementary rules. As used
herein "another nucleic acid" may refer to a separate molecule or a
spatial separated sequence of the same molecule.
[0087] As used herein, the term "complementary" or "complement(s)"
also refers to a nucleic acid comprising a sequence of consecutive
nucleobases or semi-consecutive nucleobases (e.g., one or more
nucleobase moieties are not present in the molecule) capable of
hybridizing to another nucleic acid strand or duplex even if less
than all the nucleobases do not base pair with a counterpart
nucleobase. In certain embodiments, a "complementary" nucleic acid
comprises a sequence in which about 70%, about 71%, about 72%,
about 73%, about 74%, about 75%, about 76%, about 77%, about 77%,
about 78%, about 79%, about 80%, about 81%, about 82%, about 83%,
about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,
about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99%, to about 100%, and any
range derivable therein, of the nucleobase sequence is capable of
base-pairing with a single or double stranded nucleic acid molecule
during hybridization. In certain embodiments, the term
"complementary" refers to a nucleic acid that may hybridize to
another nucleic acid strand or duplex in stringent conditions, as
would be understood by one of ordinary skill in the art.
[0088] In certain embodiments, a "partly complementary" nucleic
acid comprises a sequence that may hybridize in low stringency
conditions to a single or double stranded nucleic acid, or contains
a sequence in which less than about 70% of the nucleobase sequence
is capable of base-pairing with a single or double stranded nucleic
acid molecule during hybridization.
[0089] These definitions generally refer to a single-stranded
molecule, but in specific embodiments will also encompass an
additional strand that is partially, substantially or fully
complementary to the single-stranded molecule. Thus, a nucleic acid
may encompass a double-stranded molecule or a triple-stranded
molecule that comprises one or more complementary strand(s) or
"complement(s)" of a particular sequence comprising a molecule. As
used herein, a single stranded nucleic acid may be denoted by the
prefix "ss," a double stranded nucleic acid by the prefix "ds," and
a triple stranded nucleic acid by the prefix "ts."
[0090] C. Solid Support and Devices
[0091] A solid support is a structure containing material that will
reversibly bind with nucleic acids, particularly small RNA
molecules, and in some embodiments, it will not bind one or more
other types of macromolecules in the sample. Material may comprise
plastic, glass, silica, a magnet, a metal such as gold, carbon,
cellulose, latex, polystyrene, and other synthetic polymers, nylon,
cellulose, nitrocellulose, polyacrylate, polyacrylonitrile,
methacrylate, and/or methyl methacrylate polymethacrylate,
polyvinylchloride, styrene-divinylbenzene, or any
chemically-modified plastic. They may also be porous or non-porous
materials. The structure may also be a particle of any shape that
allows the small RNA molecules to be isolated, depleted, or
separated. In some embodiments, it is a column that includes any of
the materials described above through which a lysate may be
passed.
[0092] Other components include isolation apparatuses such as
filtration devices, including spin filters or spin columns. It may
be a sphere, such as a bead, or a rod, or a flat-shaped structure,
such as a plate with wells. The structure and sample containing the
desired RNA molecules may be centrifuged, filtered, dialyzed,
and/or otherwise isolated.
[0093] When the structure is centrifuged it may be pelleted or
passed through a centrifugible filter apparatus.
[0094] The structure may also go through an additional capture
step. In some embodiments, the sample is subsequently filtered
after passage through a capture structure. The capture step can
include filtration using a pressure-driven system or gravity-based
system (for example, centrifugation). Many such structures are
available commercially and may be utilized herewith. Other examples
can be found in WO 86/05815, WO90/06045, U.S. Pat. No. 5,945,525,
all of which are specifically incorporated by reference.
II. Characterization and Quantitation of Isolated Small RNA
Molecules
[0095] Small RNA molecules obtained from samples may be analyzed or
quantitated by various methods to characterize them, quantitate
them, or use them for analysis of other biological samples.
Provided herein are methods of quantitating or analyzing RNA, or
manipulating the RNA for use in assays involving other biological
material. General methods for quantitating or analyzing RNA may be
found in Sambrook et al. (2001) or Maniatis et al. (1990). Below
are provides examples of for using small RNA molecules from
samples, however, these examples and are not meant to be
limiting.
[0096] A. Nuclease Protection Assays
[0097] Nuclease protection assays (NPAs), including both
ribonuclease protection assays (RPAs) and S1 nuclease assays, are
an extremely sensitive method for the detection, quantitation and
mapping of specific RNAs in a complex mixture of total cellular
RNA. The basis of NPAs is a solution hybridization of a
single-stranded, discrete sized antisense probe(s) to an RNA
sample. The small volume solution hybridization is far more
efficient than more common membrane-based hybridization, and can
accommodate up to 100 .mu.g of total or poly(A) RNA. After
hybridization, any remaining unhybridized probe and sample RNA are
removed by digestion with a mixture of nucleases. Then, in a single
step reaction, the nucleases are inactivated and the remaining
probe:target hybrids are precipitated. These products are separated
on a denaturing polyacrylamide gel and are visualized by
autoradiography. If nonisotopic probes are used, samples are
visualized by transferring the gel to a membrane and performing
secondary detection.
[0098] Such techniques are well known to those of ordinary skill in
the art. Commercial kits for such assays are readily available,
such as the Direct Protect.TM. Lysate RPA Kit, HybSpeed.TM. RPA
Kit, and RPA II and RPA III.TM. Ribonuclease Protection Assay Kits
from Ambion.
[0099] B. Denaturing Agarose Gel Electrophoresis
[0100] Small RNA molecules isolated from a sample may be
quantitated by gel electrophoresis using a denaturing gel system.
Acrylamide gels are the preferred matrix for separations of this
size, although high concentrations (.about.4%+) of modified agarose
such as NuSieve (FMC, 191 Thomaston St., Rockland, Me. 04841) can
also be used. A positive control should be included on the gel so
that any unusual results can be attributed to a problem with the
gel or a problem with the RNA under analysis. RNA molecular weight
markers, an RNA sample known to be intact, or both, can be used for
this purpose. It is also a good idea to include a sample of the
starting RNA that was used in the enrichment procedure. The
presence of specific small RNAs can be determined by blotting the
contents of these gels onto hybridization membranes and probing
with radioactive oligonucleotide (RNA or DNA-based) probes.
[0101] C. Assessing RNA Yield by UV Absorbence
[0102] The concentration and purity of RNA can be determined by
diluting an aliquot of the preparation (usually a 1:50 to 1:100
dilution) in TE (10 mM Tris-HCl pH 8, 1 mM EDTA) or water, and
reading the absorbence in a spectrophotometer at 260 nm and 280
nm.
[0103] An A.sub.260 of 1 is equivalent to 40 .mu.g RNA/ml. The
concentration (.mu.g/ml) of RNA is therefore calculated by
multiplying the A.sub.260 X dilution factor X 40 .mu.g/ml. The
following is a typical example:
[0104] The typical yield from 10 .mu.g total RNA is 3-5 .mu.g. If
the sample is re-suspended in 25 .mu.l, this means that the
concentration will vary between 120 ng/gl and 200 ng/.mu.l. One
.mu.l of the prep is diluted 1:50 into 49 .mu.l of TE. The
A.sub.260=0.1. RNA concentration=0.1.times.50.times.40 .mu.g/ml=200
.mu.g/ml or 0.2 .mu.g/pl. Since there are 24 .mu.l of the prep
remaining after using 1 .mu.l to measure the concentration, the
total amount of remaining RNA is 24 .mu.l X 0.2 .mu.g/.mu.l=4.8
.mu.g.
[0105] D. Other Uses of Small RNA Molecules from Samples
[0106] Small RNA molecules obtained from a sample may be analyzed
by or used in microarray technology. For example an arrays such as
a gene array are solid supports upon which a collection of
gene-specific probes has been spotted at defined locations. The
probes localize complementary labeled targets from a nucleic acid
sample, such as an RNA sample, population via hybridization. One of
the most common uses for gene arrays is the comparison of the
global expression patterns of an RNA population. Typically, RNA
isolated from two or more tissue samples may be used. The RNAs are
reverse transcribed using labeled nucleotides and target specific,
oligodT, or random-sequence primers to create labeled cDNA
populations. The cDNAs are denatured from the template RNA and
hybridized to identical arrays. The hybridized signal on each array
is detected and quantified. The signal emitting from each
gene-specific spot is compared between the populations. Genes
expressed at different levels in the samples generate different
amounts of labeled cDNA and this results in spots on the array with
different amounts of signal.
[0107] The direct conversion of RNA populations to labeled cDNAs is
widely used because it is simple and largely unaffected by
enzymatic bias. However, direct labeling requires large quantities
of RNA to create enough labeled product for moderately rare targets
to be detected by array analysis. Most array protocols recommend
that 2.5 g of polyA or 50 g of total RNA be used for reverse
transcription (Duggan, 1999). For practitioners unable to isolate
this much RNA from their samples, global amplification procedures
have been used.
[0108] The most often cited of these global amplification schemes
is antisense RNA (aRNA) amplification (U.S. Pat. Nos. 5,514,545 and
5,545,522). Antisense RNA amplification involves reverse
transcribing RNA samples with an oligo-dT primer that has a
transcription promoter such as the T7 RNA polymerase consensus
promoter sequence at its 5' end. First strand reverse transcription
creates single-stranded cDNA. Following first strand cDNA
synthesis, the template RNA that is hybridized to the cDNA is
partially degraded creating RNA primers. The RNA primers are then
extended to create double-stranded DNAs possessing transcription
promoters. The population is transcribed with an appropriate RNA
polymerase to create an RNA population possessing sequence from the
cDNA. Because transcription results in tens to thousands of RNAs
being created from each DNA template, substantive amplification can
be achieved. The RNAs can be labeled during transcription and used
directly for array analysis, or unlabeled aRNA can be reverse
transcribed with labeled dNTPs to create a cDNA population for
array hybridization. In either case, the detection and analysis of
labeled targets are well known in the art. Other methods of
amplification that may be employed include, but are not limited to,
polymerase chain reaction (referred to as PCR.TM.; see U.S. Pat.
Nos. 4,683,195, 4,683,202 and 4,800,159, and Innis et al., 1988);
and ligase chain reaction ("LCR"), disclosed in European
Application No. 320 308, U.S. Pat. Nos. 4,883,750, 5,912,148. Qbeta
Replicase, described in PCT Application No. PCT/US87/00880, may
also be used as an amplification method Alternative methods for
amplification of a nucleic acid such as RNA are disclosed in U.S.
Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,
5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,
5,928,906, 5,932,451, 5,935,825, 5,939,291, 5,916,779 and
5,942,391, GB Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025, PCT Application WO 89/06700, PCT Application WO
88/10315, European Application No. 329 822, Kwoh et al., 1989;
Frohman, 1994; Ohara et al., 1989; and Walker et al., 1992 each of
which is incorporated herein by reference in its entirety. cDNA
libraries may also be constructed and used to analyze to the RNA
extracted from a sample. Construction of such libraries and
analysis of RNA using such libraries may be found in Sambrook et
al. (2001); Maniatis et al. (1990); Efstratiadis et al. (1976);
[0109] Higuchi et al. (1976); Maniatis et al. (1976); Land et al.
(1981); Okayama et aL (1982);
[0110] Gubler et al. (1983); Ko (1990); Patanjali et al. (1991);
U.S. Patent Appln. 20030104468, each incorporated herein by
reference.
[0111] The present methods and kits may be employed for high volume
screening. A library of RNA or DNA can be created using methods and
compositions of the invention. This library may then be used in
high throughput assays, including microarrays. Specifically
contemplated by the present inventors are chip-based nucleic acid
technologies such as those described by Hacia et al. (1996) and
Shoemaker et aL (1996). Briefly, these techniques involve
quantitative methods for analyzing large numbers of genes rapidly
and accurately. By using fixed probe arrays, one can employ chip
technology to segregate target molecules as high density arrays and
screen these molecules on the basis of hybridization (see also,
Pease et aL, 1994; and Fodor et al, 1991). The term "array" as used
herein refers to a systematic arrangement of nucleic acid. For
example, a nucleic acid population that is representative of a
desired source (e.g., human adult brain) is divided up into the
minimum number of pools in which a desired screening procedure can
be utilized to detect or deplete a target gene and which can be
distributed into a single multi-well plate. Arrays may be of an
aqueous suspension of a nucleic acid population obtainable from a
desired mRNA source, comprising: a multi-well plate containing a
plurality of individual wells, each individual well containing an
aqueous suspension of a different content of a nucleic acid
population. Examples of arrays, their uses, and implementation of
them can be found in U.S. Pat. Nos. 6,329,209, 6,329,140,
6,324,479, 6,322,971, 6,316,193, 6,309,823, 5,412,087, 5,445,934,
and 5,744,305, which are herein incorporated by reference.
[0112] Microarrays are known in the art and consist of a surface to
which probes that correspond in sequence to gene products (e.g.,
cDNAs, mRNAs, cRNAs, polypeptides, and fragments thereof), can be
specifically hybridized or bound at a known position. In one
embodiment, the microarray is an array (i.e., a matrix) in which
each position represents a discrete binding site for a product
encoded by a gene (e.g., a protein or RNA), and in which binding
sites are present for products of most or almost all of the genes
in the organism's genome. In a preferred embodiment, the "binding
site" (hereinafter, "site") is a nucleic acid or nucleic acid
analogue to which a particular cognate cDNA can specifically
hybridize. The nucleic acid or analogue of the binding site can be,
e.g., a synthetic oligomer, a full-length cDNA, a less-than full
length cDNA, or a gene fragment.
[0113] The nucleic acid or analogue are attached to a solid
support, which may be made from glass, plastic (e.g.,
polypropylene, nylon), polyacrylamide, nitrocellulose, or other
materials. A preferred method for attaching the nucleic acids to a
surface is by printing on glass plates, as is described generally
by Schena et al., 1995a. See also DeRisi et al., 1996; Shalon et
al., 1996. Other methods for making microarrays, e.g., by masking
(Maskos et al., 1992), may also be used. In principal, any type of
array, for example, dot blots on a nylon hybridization membrane
(see Sambrook et al., 2001, which is incorporated in its entirety
for all purposes), could be used, although, as will be recognized
by those of skill in the art, very small arrays will be preferred
because hybridization volumes will be smaller.
[0114] Use of a biochip is also contemplated, which involves the
hybridization of a labeled molecule or pool of molecules to the
targets immobilized on the biochip.
III. Kits
[0115] In further embodiments of the invention, there is a provided
a kit for the isolation of small RNA molecules, such as miRNA and
siRNA from a sample, particularly a cell sample. Any of the
compositions described herein may be comprised in a kit. In a
non-limiting example, reagents for lysing cells, extracting RNA the
cell lysate, and/or analyzing or quantitating the RNA obtained may
be included in a kit. The kits will thus comprise, in suitable
container means, any of the reagents disclosed herein. It may also
include one or more buffers or solutions, such as lysis buffer,
extraction buffer, solutions to have alcohol added, elution
solution, wash solution and other components for isolating the
desired RNA, such as a capture structure. In some embodiments,
there are kits for isolating small RNA molecules comprising: a)
Acid Phenol-Chloroform; b) Lysis/Binding Buffer (GuSCN-based); c)
miRNA Homogenate Additive (2M Sodium Acetate, pH 4, to be added in
0.1 vol before extraction with PC); d) miRNA Wash Soln #1 (1.6M
GuSCN in 70% ethanol); e) Wash Soln #2/3 (80% ethanol, 0.1 M NaCl,
4.5 mM EDTA, 10 mM TrisHCl, pH 7.5); f) Elution Solution (0.1 mM
EDTA, pH8); g) Gel Loading Buffer II; h) Collection Tubes; i)
Filter Cartridges.
[0116] The components of the kits may be packaged either in aqueous
media or in lyophilized form. The container means of the kits will
generally include at least one vial, test tube, flask, bottle,
syringe or other container means, into which a component may be
placed, and preferably, suitably aliquoted. Where there are more
than one component in the kit (they may be packaged together), the
kit also will generally contain a second, third or other additional
container into which the additional components may be separately
placed. However, various combinations of components may be
comprised in a vial. The kits of the present invention also will
typically include a means for containing the RNA, and any other
reagent containers in close confinement for commercial sale. Such
containers may include injection or blow-molded plastic containers
into which the desired vials are retained. When the components of
the kit are provided in one and/or more liquid solutions, the
liquid solution is an aqueous solution, with a sterile aqueous
solution being particularly preferred.
[0117] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means. The container means will
generally include at least one vial, test tube, flask, bottle,
syringe and/or other container means, into which the nucleic acid
formulations are placed, preferably, suitably allocated. The kits
may also comprise a second container means for containing a
sterile, pharmaceutically acceptable buffer and/or other
diluent.
[0118] Such kits may also include components that preserve or
maintain the RNA or that protect against its degradation. Such
components may be RNAse-free or protect against RNAses. Such kits
generally will comprise, in suitable means, distinct containers for
each individual reagent or solution.
[0119] A kit will also include instructions for employing the kit
components as well the use of any other reagent not included in the
kit. Instructions may include variations that can be
implemented.
EXAMPLES
[0120] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
[0121] Preparation of Cell Lysate and Isolation of RNA
[0122] The following procedure provides the basis for the invention
and is referred to in the Examples as the Ambion miRNA Isolation
Kit (AMIK) procedure.
[0123] Frozen tissue was ground under liquid nitrogen to a fine
powder. Lysis buffer (4 M GuSCN; 0.1 M beta-mercaptoethanol; 0.5%
N-lauroyl sarcosine; 25 mM Na-citrate, pH 7.2) was added to this
powder in an appropriate vessel at a proportion of 1 ml to every
gram of tissue powder. This was homogenized using mechanical means
to create a finely-dispersed tissue lysate. One tenth volume of a 2
M Na acetate (pH 4.0) solution was added and mixed thoroughly,
adding 0.1 ml for every ml. The lysate was then processed
immediately (without organic extraction) or placed on ice to be
processed within 15 minutes.
[0124] Processing involved the addition of an equal volume of Acid
Phenol-Chloroform (5:1, equilibrated with aqueous solution at pH
4.5) to the suspension, followed by vigorous agitation (by
vortexing or shaking) for 30-60 sec. The phenol-chloroform and
aqueous phases were then separated by centrifugation at
16,000.times.G for 5 min, or until a clear interface was obtained.
The aqueous phase was removed by aspiration, avoiding withdrawing
any of the interface between phases. This aqueous phase, which
contained the RNA from the sample, was made into a concentration of
55% ethanol by addition of 1.22 volumes of ethanol.
[0125] Immediately after mixing, the sample was applied directly to
a glass fiber column, as used in an RNAqueous kit.RTM.(Ambion). The
sample was passed through the filter by centrifugation at
.about.12,000.times.G for 1 min, then the filter was washed by the
successive passage of three wash solutions through it. The
collection tube was emptied between each wash, and each wash was
passed completely through the filter at .about.12,000.times.G for 1
min or longer, if required to pass all fluid. The first wash was
with 0.5 ml of 1.6 M guanidinium isocyanate (GuSCN)/70% ethanol,
the last two with 80% alcohol/0.1 M NaCl/4.5 mM EDTA/10 mM TrisHCl,
pH 7.5. After the last wash was passed through the filter, the
filter was re-centrifuged over an empty collection tube to remove
all traces of ethanol.
[0126] The sample was then eluted off the filter with 100 .mu.l of
0.1 mM EDTA, pH 8.0, which was applied directly to the filter at
room temperature and centrifuged through into a fresh collection
tube. FIG. 1 and FIG. 2 show the differences between preparations
made from three different tissues, heart, brain, and liver, without
and with the the pre-extraction step. It can be seen that, in
either circumstance, a substantial portion of the let-7 miRNA is
captured at 55% ethanol.
Example 2
[0127] Detection of miRNAs through Northern Blotting
[0128] For each RNA sample, 5 .mu.l was combined with 5 .mu.l of
Gel Loading Dye II (Ambion). Prior to loading on a denaturing
acrylamide gel, these samples were heated at 95.degree. C. for 2-5
minutes. The standard gel was 15% acrylamide (monomer:bis ratio of
19:1), 7M urea, buffered with TBE (Tris-Borate-EDTA, Peacock and
Dingman, 1967).
[0129] The gel was routinely pre-run at 300-450 V for 30 minutes
prior to loading the samples in sample buffer, which also contained
bromphenol blue and xylene cyanol tracking dyes. The
electrophoresis was performed at 300-450 V for 45-60 min, or until
the bromphenol blue tracking dye was in the lower quadrant of the
gel.
[0130] After electrophoresis, the gel apparatus was disassembled
and the gel was electroblotted to a BrightStar-Plus Nylon membrane
(Ambion). This procedure can be performed in a semi-dry apparatus
using a stack of three sheets of Whatman filter paper (3MM) soaked
in 0.25.times.TBE above and below the gel sandwich at 200-400 mA
for at least 0.2 A-hr. Extending this time does not lose sample.
After blotting, the membrane was kept damp and UV crosslinked using
a commercial crosslinking device (the Stratalinker.TM., Stratagene,
Inc.)
[0131] The membrane was probed for the specific miRNA, let-7
(Pasquinelli et al., 2000) using an antisense probe that was 5'
end-labeled by T4 Polynucleotide kinase.
[0132] In some cases other ubiquitous small RNAs were also probed
for with antisense oligodeoxyribonucleotides at the same time.
These included the U2 snRNA (Accession # X07913, complementary to
the positions 28-42 of the 187 nt mouse U2 snRNA), U6 snRNA
(Accession # V00853 or J00648, complementary to positions 83-103 of
the 106 nt mouse RNA), and U43 snoRNA (Accession # AJ238853,
complementary to positions 20-37 of the 62 nt human U43 RNA). All
of these cross-hybridize readily between mouse and human. The
procedure of Patterson and Guthrie (1987) was followed for
prehybridization, hybridization, and washing (Patterson and
Guthrie, 1987). The blots were prehybridized in {6.times.SSC,
10.times.Denhardt's solution, 0.2% SDS} at 65.degree. C. for at
least one hour, then 10 ml of hybridization solution added
{6.times.SSC, 5.times. Denhardt's, 0.2% SDS} which contained 5'
end-labeled let-7, U43, U6, and/or U2 antisense
oligodeoxynucleotide probes (U43, U6, let-7 minimum=400,000 cpm ;
U2 minimum=200,000 cpm) and had been filtered (0.45 .mu.m pore)
prior to use. Hybridization was for 8-24 hr with agitation at room
temperature. After hybridization, solutions were removed, and the
blot washed 3 times for 5 minutes at RT with wash solution:
{6.times.SSC, 0.2% SDS}, then once with the same wash solution at
42.degree. C. After the final wash, the blots were wrapped in
plastic wrap and exposed to a phosphorimager screen (Molecular
Dynamics) as per the manufacturer's instructions to quantify the
amount of signal present in each band. The amount of let-7 in the
fraction eluted was often compared to that in the flow-through,
providing a "% bound" figure, as given in FIG. 1, FIG. 2, FIG. 3,
FIG. 4, FIG. 5, and FIG. 6. For other figures, the amount of let-7
was compared to other samples or given as absolute. See specific
examples.
[0133] For some of the examples, a second Northern blot was made
from an agarose gel system to look for the presence of larger RNA
species. These were mRNAs for the ubiquitously-expressed genes
cyclophilin (=Peptidylproline Isomerase or PPI), GAPDH and/or
.beta.-actin. The agarose gels were run and blotted using the
NorthernMax Gly kit as described by Ambion. For probing the blot,
antisense RNA probes were transcribed from templates supplied by
Ambion (cat#'s 7675, 7431, 7423) and hybridized in Ultrahyb
(Ambion), using all protocols as specified in Ambion
literature.
Example 3
[0134] Enrichment of Small RNA Molecules
[0135] Frozen mouse brain, heart, liver, and kidney were processed
separately according to the following protocol for enrichment of
small RNAs.
[0136] Approximately one-half gram of frozen mouse (strain
Swiss-Webster, 6-12 weeks old) tissue was crushed to fine powder
under liquid nitrogen in a mortar. This powder was further
dispersed in standard lysis buffer (4 M GuSCN; 0.1 M
beta-mercaptoethanol; 0.5% N-lauroyl sarcosine; 25 mM Na-citrate,
pH 7.2) by the use of a rotor-stator homogenizer with a 7 mm
generator at high speed for .about.30 sec.
[0137] After homogenization, 0.6 ml of the lysate was removed for
this study. 60 .mu.l of 2M Na-acetate, pH 4.0, was added to the
lysate, followed immediately by 0.6 ml of acid phenol-chloroform.
After 30 sec of vigorous agitation, the aqueous phase was separated
by centrifugation at 16,000.times.G for 5 min. Four 100 .mu.l
aliquots of this aqueous phase were used in four separate
separations. The four aliquots had 100 .mu.l of 40%, 50%, 60%, and
70% ethanol added to each, then were passed through glass fiber
filters as in the RNAqueous procedure (Ambion). The 20%, 25%, 30%,
and 35% ethanol solutions that passed through these filters (the
flow-through) was then adjusted to 55% ethanol final concentration
by the addition of 156, 133, 111, and 88.9 .mu.l of ethanol,
respectively. All four samples were passed over separate glass
fiber filter columns. The filters were then washed with 0.7 ml of 4
M guanidinium isocyanate (GuSCN)/70% ethanol, followed by two
washes with 0.5 ml 80% alcohol/0.1 M NaCl/4.5 mM EDTA/10 mM
TrisHCl, pH 7.5. After each wash was passed through the filter, the
collection tube was emptied and replaced. Each wash was passed
through the filter by centrifugation as per the RNAqueous protocol
(Ambion). filter re-centrifuged over an empty collection tube to
remove all traces of ethanol. The sample was then eluted off the
filter with 100 .mu.l of 0.1 mM EDTA, pH 8.0, which was applied
directly to the filter at room temperature and centrifuged through
into a fresh collection tube. The samples were examined by Northern
blot, as described, and compared on the same gel to another sample
that had been prepared from an equal volume of the same lysate
using the Totally RNATM kit from Ambion.
[0138] Using both agarose and acrylamide Northern blots, the levels
of the .beta.-actin, GAPDH, U2, and let-7 RNA species present in
frozen mouse brain, heart, liver, and kidney were assayed in the
material eluted from the first and second columns to determine the
fraction recovered in the latter. These are shown on FIG. 7. The
larger mRNA is completely removed from the small-RNA enriched
fraction.
[0139] FIG. 8 shows the relative enrichment of small RNAs using the
method described in Example 3 as compared to the standard RNA
isolation method. Here, samples of four common mouse tissues:
brain, heart, kidney, and liver, were homogenized in standard lysis
buffer as described in Example 1. After homogenization, two equal
aliquots were taken of each lysate. One was subjected to a standard
RNA preparative procedure using organic extraction and ethanol
precipitation, using 4 volumes of ethanol to precipitate to ensure
full recovery of small RNA species. The other aliquot was subjected
to the enrichment procedure as described in Example 3. The
concentration of RNA in each final sample was quantified using
absorbence at 260 nm. One microgram of each sample was separated on
a 15% denaturing polyacrylamide gel. This gel was electroblotted
and the resultant Northern blot probed for let-7 and U2as described
in Example 2. The amount of each probe hybridized to the
appropriate area of the blot was used to determine the relative
amounts of each RNA in the 1 .mu.g samples. The signal for the
enriched samples was divided by the signal for the standard samples
to provide the enrichment factors given in FIG. 8. Enrichment in
this case was from .about.3.5-8-fold by mass.
Example 4
[0140] Comparison to Standard Organic Extraction and Ethanol
Precipitation
[0141] Samples from two mouse livers that had been stored frozen at
-80.degree. C. were ground to a fine powder under liquid nitrogen
and homogenized in 10 volumes (ml/g) the standard lysis buffer (4 M
GuSCN; 0.1 M .beta.-mercaptoethanol; 0.5% N-lauroyl sarcosine; 25
mM Na-citrate, pH 7.2) and then divided into four aliquots. One of
the aliquots was extracted twice with two different
phenol-chloroform solutions as described in the Totally RNA.TM.
protocol (Ambion), and the other three were subjected individually
to the standard AMIK procedure. The RNA pelleted from the Totally
RNA.TM. procedure was redissolved in 100 .mu.l of 0.1 mM EDTA, pH
8. The final elution for the AMIK samples was in the same volume
and same solution. Samples were electrophoresed and blotted as
described on both 15% acrylamide and 1% agarose gels. The
appropriate blots were probed for .beta.-actin, GAPDH, U2, U43, and
let-7 as described. The recoveries of each RNA relative to the
extraction procedure are summarized in the graph in FIG. 9. The
yield from the invention generated amounts of small RNAs equal to
or greater than the organic extraction procedure.
Example 5
[0142] Comparison to Standard Glass-Fiber Filter Purification
[0143] Frozen mouse liver and frozen mouse brain samples stored at
-80.degree. C. were homogenized into standard lysis buffer at a
ratio of 1 g tissue to 10 ml buffer. After homogenization, all
lysates were stored on ice until one of two processing procedures
was applied.
[0144] Starting with six aliquots of 100 .mu.l from each parent
lysate, 2 samples were processed by the RNeasy method from Qiagen,
following their mini procedure precisely after addition of 250
.mu.l of the Tissue Lysis Buffer (TLB) supplied with the kit. The
final four aliquots from each tissue were prepared by the AMIK
method previously described. The samples were all eluted in 100
.mu.l of water. For analysis, 5 .mu.l of each of the samples were
analyzed by electrophoresis on 15% acrylamide gels and blotting,
and the blots were probed for let-7, U43, and U2. After using
phosphorimagery to quantify the bands, the signal levels were
compared between the methods for each small RNA. These results are
shown in FIG. 10. This invention was much more efficient at
capturing all small RNAs than the standard glass-fiber filter
extraction procedure. This inability to capture small molecules
with a standard procedure is affected to some extent by the type of
tissue as well, since the capture from liver lysate appears to be
more efficient. This observation is consistent with our
observations using raw lysate (FIG. 1).
Example 6
[0145] Efficiency of Small RNA Recovery from Raw Lysate in Three
Different Tissues.
[0146] Samples of frozen heart, liver, and brain from mice (strain
Swiss-Webster, 6-12 weeks old) were each pulverized under liquid
nitrogen to a powder. This powder was weighed frozen and 10 ml of
lysis buffer per gram of tissue was added (weights ranged from 200
to 500 mg). Samples were homogenized with a rotor-stator
homogenizer immediately after addition, then divided into
8.times.100 .mu.l aliquots on ice. To these, 53.9, 66.7, 81.8, 100,
122.2, 150, 185.7, and 233.3 .mu.l of absolute ethanol were added
to make final concentrations of 35, 40, 45, 50, 55, 60, 65, and 70%
ethanol. Each of these was passed over a glass fiber filter column
as found in the RNAqueous.RTM. kit (Ambion), and the flow-through
from this collected. The RNA in the flow-through was
phenol-chloroform extracted and ethanol precipitated with four
volumes of ethanol to ensure precipitation of small RNAs. After
pelleting the RNA by 30 min of centrifugation at 16,000.times.G,
the pellet was washed once with 80% ethanol and then redissolved in
60 .mu.l of 0.5 mM EDTA, pH 8.0. The filters were washed three
times, once with 0.7 ml of 4 M guanidinium isocyanate (GuSCN)/70%
ethanol, followed by two washes with 0.5 ml 80% alcohol/0.1 M
NaCl/4.5 mM EDTA/10 mM TrisHCI, pH 7.5. Each wash was performed as
above, by centrifugation at 12,000.times.G for 1 min or sufficient
time to clear all liquid through the filter, with collection tubes
emptied after each. Samples were eluted using 2 separate additions
of 30 .mu.l of 0.1 mM EDTA, pH 8.0 which was applied directly to
the filter after pre-warming to 95.degree. C., each centrifuged
through into the same fresh collection tube. Equal amounts (5
.mu.l) of both the filter-bound-and-eluted and the flow-through
were analyzed by Northern blot as described above. Since bound and
flow-through were on the same blot, the amount of let-7 RNA bound
could be calculated for each ethanol concentration with each
tissue. This data is plotted in FIG. 1 and FIG. 2. It is apparent
that the binding behavior for each tissue was different, in terms
of the concentration of ethanol required to immobilize all let-7
RNA on the glass fiber filter. However, the maximum appears to be
achieved for all tissues by 55% ethanol.
Example 7
[0147] Purification from Cultured Cells
[0148] Cells were collected from two lines, HEK-293 (derived from
human embryonic kidney) or HeLa (human cervix) cells, from culture
flasks by trypsinization. After counting, these cells were added at
a level of about one million each to two 2 ml microcentrifuge tubes
and pelleted by centrifugation. Supernatant was removed and the
pelleted contents of each tube was resuspended in 700 .mu.l of
lysis buffer as described in the standard procedure (Example 1).
The cells were lysed by agitating the tube vigorously for 30 sec
rather than use of a homogenization apparatus. For each set, one
set was immediately made about 55% ethanol by addition of 860 .mu.l
absolute ethanol. The other aliquot was processed as stated in the
standard procedure: acidified by the addition of 70 .mu.l 2 M
Na-Acetate buffered to pH 4, followed by extraction with 700 .mu.l
acid phenol-chloroform, then addition of 860 .mu.l ethanol to the
recovered upper phase. Both samples were passed through glass-fiber
filters, washed three times, and eluted with 100 .mu.l 0.1 mM EDTA,
pH 8 as described above. Five .mu.l of each eluate was
electrophoresed on a 15% acrylamide gel and Northern blotted for U2
and let-7. The levels of each, as determined from phosphorimagery
of the blot, are shown in FIG. 11. The recovery of small RNAs from
all the methods appears good, but recovery from HeLa cells was
enhanced by the pre-extraction procedure.
Example 8
[0149] Pre-extraction Using Different Salt Conditions
[0150] Frozen mouse liver was homogenized into Lysis Buffer at
1.1.times. normal concentration minus Na-citrate (4.4 M GuSCN; 0.11
M .beta.-mercaptoethanol; 0.55% N-lauroyl sarcosine). Immediately
after homogenization, two 1.8 ml aliquots were removed from this
lysate and 200 .mu.l of 0.25M Na-citrate at either pH 7.2 or 4.5
was added to each. Four 400 .mu.l aliquots were removed from these
2 ml portions, and 40 .mu.l of either water, 1M, 2M, or 3M
NaOOCCH.sub.3 (sodium acetate, pH 4.5) was added to each of these,
to give a final [NaOOCCH.sub.3] of zero and about 0.1, 0.2, and 0.3
M. The samples were each extracted with 440 .mu.l of acid
phenol-chloroform and 300 .mu.l of the upper phase recovered. This
was made 55% in ethanol by the addition of absolute ethanol and
purified over a glass-fiber filter column as described in the
standard procedure. Each sample was applied to a 15% acrylamide
gel, blotted and probed as described above. The levels of U2, U43,
and let-7 determined for each are shown in the FIG. 12 graph. Yield
appears to be roughly equivalent at both pH's (although U43 was
variable), but the best yield appears in the presence of 0.2 M
NaOOCCH.sub.3 at both pH's.
Example 9
[0151] Binding of Small RNA Molecules at Different Guanidinium and
Ethanol Concentrations
[0152] Mouse liver was homogenized in standard lysis buffer and
extracted with acid phenol-chloroform. The extracted lysate was
divided in two portions. An equal volume was added to each
consisting of either Lysis Buffer with no guanidinium (0.1 M
.beta.-mercaptoethanol; 0.5% N-lauroyl sarcosine; 25 mM Na-citrate,
pH 7.2) or Lysis Buffer with 2 M GuSCN (2 M GuSCN; 0.1 M
beta-mercaptoethanol; 0.5% N-lauroyl sarcosine; 25 mM Na-citrate,
pH 7.2), creating solutions with a final [GuSCN] of 2 M and 3 M,
respectively. These were then further divided into 18 aliquots of
200 .mu.l each, and ethanol additions made in one of two manners.
The first method was the addition of 22.2, 35.3, 50, 66.7, 85.7,
107.7, 133.3, and 200 .mu.l of absolute ethanol. This gave final
ethanol concentration of 10, 15, 20, 25, 30, 35, 40, 45, and 50%,
with corresponding final guanidinium concentrations that decreased
with increasing ethanol. (2.7, 2.55, 2.4, 2.25, 2.1, 1.95, 1.8,
1.65, and 1.5 M for 3 M initial concentration; 1.8, 1.7, 1.6, 1.5,
1.4, 1.3, 1.2, 1.1, and 1.0 M for 2 M initial concentration). The
second method added equal volumes of ethanol solutions in water
from 20-100% to give the same final ethanol concentrations, but
with consistent guanidinium concentrations of 1.5 or 1 M within
each series. After ethanol addition, each of these samples was
bound to the glass-fiber filter and the standard procedure was
followed. Samples were run on both acrylamide and agarose gels to
assay for the presence of .beta.-actin, GAPDH, PPI (cyclophilin),
U2, and let-7. The binding behavior of each species as ethanol
concentration increased was plotted for the four series in FIG. 3,
FIG. 4, FIG. 5, and FIG. 6. From these series, it is demonstrated
that differences exist in the behavior of the differently-sized RNA
species, such that by manipulating both salt and ethanol
concentration the binding of quite restricted size ranges of RNA
molecules can be achieved, indicating more refined
size-fractionation procedures can be performed.
Example 10
[0153] Use of Small RNAs to Probe Microarrays
[0154] Small RNAs enriched using procedures described in Examples 3
or 9 may be used in the microarray technologies described in the
specification. In one example, the probes affixed to the microarray
may contain sequences specifically designed to capture known miRNAs
or siRNAs. Alternatively, the probes affixed to the microarray
could be mRNA sequences to look for potential in vivo biological
targets for miRNAs or siRNAs. The small RNA molecule population
could be labeled radioactively or with tags that are reactive to
light or able to bind secondary molecules capable of reacting with
light. These direct or indirect labels could be attached through
enzymatic means well-known to those of skill in the art such as:
removal of the 5' phosphate with phospahtase followed by addition
of modified phosphate with polynucleotide kinase; or addition to
the 3' end of one or several tagged nucleotides with RNA ligase or
polymerases such as poly-A polymerase.
[0155] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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