U.S. patent application number 11/363982 was filed with the patent office on 2007-08-30 for methods and compositions for the rapid isolation of small rna molecules.
This patent application is currently assigned to Sigma-Aldrich Co.. Invention is credited to Fuqiang Chen, Carol Kreader.
Application Number | 20070202511 11/363982 |
Document ID | / |
Family ID | 38444449 |
Filed Date | 2007-08-30 |
United States Patent
Application |
20070202511 |
Kind Code |
A1 |
Chen; Fuqiang ; et
al. |
August 30, 2007 |
Methods and compositions for the rapid isolation of small RNA
molecules
Abstract
The present invention provides extraction compositions and
methods for the rapid and efficient isolation of small RNA
molecules from a biological sample. In particular, the extraction
compositions, when contacted with a biological sample, releases the
small RNA molecules from the other molecules in a biological
sample, and the released small RNA molecules may then be
isolated.
Inventors: |
Chen; Fuqiang; (St. Louis,
MO) ; Kreader; Carol; (Kirkwood, MO) |
Correspondence
Address: |
Polsinelli Shalton Welte Suelthaus PC
Suite 1100
100 S. Fourth Street
St. Louis
MO
63102
US
|
Assignee: |
Sigma-Aldrich Co.
|
Family ID: |
38444449 |
Appl. No.: |
11/363982 |
Filed: |
February 28, 2006 |
Current U.S.
Class: |
435/6.16 ;
435/270; 536/25.4 |
Current CPC
Class: |
C12N 15/1003 20130101;
C12Q 1/6806 20130101; C12Q 1/6806 20130101; C12Q 2527/125
20130101 |
Class at
Publication: |
435/006 ;
435/270; 536/025.4 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 1/08 20060101 C12N001/08; C07H 21/02 20060101
C07H021/02 |
Claims
1. A method for isolating a small RNA from a biological sample, the
method comprising: a) contacting the biological sample with a
chaotropic agent and a metal salt, wherein the small RNA is
released from debris in the biological sample; and b) separating
the small RNA from the debris.
2. The method of claim 1, wherein the biological sample is
contacted with the chaotropic agent and the metal salt
simultaneously.
3. The method of claim 1, wherein the biological sample is
contacted with the chaotropic agent and the metal salt
sequentially.
4. The method of claim 1, wherein the chaotropic agent is selected
from the group consisting of guanidine hydrochloride, guanidine
thiocyante, guanidine carbonate, sodium perchlorate, sodium iodide,
sodium trichloroacetate, and urea.
5. The method of claim 1, wherein the metal salt is a group IA or
group IIA metal salt.
6. The method of claim 1, wherein the metal salt is a lithium
salt.
7. The method of claim 6, wherein the lithium salt is selected from
the group consisting of lithium chloride, lithium acetate, lithium
citrate, lithium carbonate, and lithium borate.
8. The method of claim 1, wherein the chaotropic agent is guanidine
hydrochloride and the metal salt is lithium chloride.
9. The method of claim 1, wherein the concentration of the
chaotropic agent is from about 1 M to about 8 M and the
concentration of the metal salt is from about 1 M to about 8 M.
10. The method of claim 1, wherein the chaotropic agent and the
metal salt are in a solution having a pH from about 3 to about
8.
11. The method of claim 1, wherein the chaotropic agent and the
metal salt are in a solution having a pH from about 3 to about
4.
12. The method of claim 1, wherein the small RNA is soluble after
the biological sample is contacted with the chaotropic agent and
the metal salt.
13. The method of claim 1, wherein the small RNA is separated from
the debris by centrifugation to form a clarified mixture.
14. The method of claim 13, wherein the small RNA is isolated from
the clarified mixture by immobilization on a chromatographic
binding matrix in the presence of a high concentration of
alcohol.
15. The method of claim 1, wherein the small RNA is separated from
the debris by chromatography.
16. The method of claim 1, wherein the biological sample is
contacted with a chaotropic agent, a metal salt, and an agent
selected from the group consisting of a detergent, a buffer, a
thiol-reducing agent, an antifoaming agent, and a bulking
agent.
17. The method of claim 1, wherein the biological sample is
selected from the group consisting of a cell, a tissue from a
multicellular organism, a whole organism, a virus, a mixture of
nucleic acids generated in vitro, and a body fluid, such as serum,
blood, urine, saliva, cerebrospinal fluid, and semen.
18. The method of claim 17, wherein the cell is derived from a host
selected from the group consisting of prokaryotes, fungi, plants,
invertebrates, and vertebrates.
19. The method of claim 1, wherein the small RNA is selected from
the group consisting of snRNA, snoRNA; miRNA, siRNA, tasiRNA,
rasiRNA, stRNA, tncRNA, scRNA, and smRNA.
20. The method of claim 1, wherein the small RNA is less than
approximately 200 nucleotides in length.
21. The method of claim 1, wherein the small RNA is less than
approximately 100 nucleotides in length.
22. The method of claim 1, wherein the small RNA is less than
approximately 30 nucleotides in length.
23. The method of claim 1, wherein the small RNA is single
stranded.
24. The method of claim 1, wherein the small RNA is double
stranded.
25. A kit for isolating a small RNA from a biological sample, the
kit comprising: a) a chaotropic agent; b) a metal salt; and b)
instructions for isolating the small RNA.
26. The kit of claim 25, wherein the metal salt is a group IA or
group IIA metal salt.
27. The kit of claim 25, wherein the metal salt is a lithium
salt.
28. The kit of claim 27, wherein the lithium salt is selected from
the group consisting of lithium chloride, lithium acetate, lithium
citrate, lithium carbonate, and lithium borate.
29. The kit of claim 25, wherein the chaotropic agent is selected
from the group consisting of guanidine hydrochloride, guanidine
thiocyante, guanidine carbonate, sodium perchlorate, sodium iodide,
sodium trichloroacetate, and urea.
30. The kit of claim 25, wherein the chaotropic agent is guanidine
hydrochloride and the metal salt is lithium chloride.
31. The kit of claim 30, wherein the concentration of the guanidine
hydrochloride is about 7 M and the concentration of the lithium
chloride is about 12 M.
32. The kit of claim 25, wherein the kit further comprises an agent
selected from the group consisting of a buffer, a detergent, a
thiol-reducing agent, an antifoaming agent, and a bulking
agent.
33. The kit of claim 25, wherein the chaotropic agent is in a
solution having a pH of about 3 to about 8.
34. The kit of claim 25, wherein the chaotropic agent is in a
solution having a pH of about 3 to about 4.
35. The kit of claim 25, wherein the kit further comprises a means
to separate the small RNA from the biological sample.
36. The kit of claim 35, wherein the separation means is a
chromatographic binding matrix selected from the group consisting
of silica, borosilicate, diatomaceous earth, aluminum oxides,
glass, titanium oxides, zirconium oxides, and hydroxyapatite.
37. The kit of claim 36, wherein the binding matrix is a filter
comprising borosilicate fibers.
38. An extraction composition comprising a chaotropic agent and a
metal salt.
39. The composition of claim 38, wherein the chaotropic agent is
selected from the group consisting of guanidine hydrochloride,
guanidine thiocyante, guanidine carbonate, sodium perchlorate,
sodium iodide, sodium trichloroacetate, and urea.
40. The extraction composition of claim 38, wherein the metal salt
is a group IA or group IIA metal salt.
41. The extraction composition of claim 38, wherein the metal salt
is a lithium salt.
42. The extraction composition of claim 41, wherein the lithium
salt is selected from the group consisting of lithium chloride,
lithium acetate, lithium citrate, lithium carbonate, and lithium
borate.
43. The extraction composition of claim 38, wherein the chaotropic
agent is guanidine hydrochloride and the metal salt is lithium
chloride.
44. The extraction composition of claim 43, wherein the
concentration of the guanidine hydrochloride present in the
extraction composition is from about 3 M to about 6 M and the
concentration of the lithium salt present in the extraction
composition is from about 1.5 M to about 6 M.
45. The extraction composition of claim 38, wherein the extraction
composition further comprises an agent selected from the group
consisting of a buffer, a detergent, a thiol-reducing agent, an
antifoaming agent, and a bulking agent.
46. The extraction composition of claim 38, wherein the pH of the
extraction composition is from about 3 to about 8.
47. The extraction composition of claim 38, wherein the pH of the
extraction composition is from about 3 to about 4.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods, compositions, and
kits to isolate small RNA molecules from biological samples.
BACKGROUND OF THE INVENTION
[0002] More than a decade ago a non-coding 22-nucleotide (nt) RNA
(lin-4) was discovered that played an important role in the
developmental timing of Caenorhabditis elegans. It was not
realized, however, until just a just few years ago that small RNA
molecules such as lin-4 are ubiquitous and play important
regulatory roles in virtually all eukaryotes. Recent work has shown
that prokaryotes and viruses also express small regulatory RNA
molecules. Thus, in addition to large RNA molecules, such as
messenger RNA (mRNA) and ribosomal RNA (rRNA), cells express an
array of small RNA molecules, including 5.8S rRNA, 5S rRNA,
transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA
(snoRNA); micro RNA (miRNA), small interfering RNA (siRNA),
trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA),
small temporary RNA (stRNA), tiny non-coding RNA (tncRNA), small
scan RNA (scRNA), and small modulatory RNA (smRNA). Micro RNA
molecules, which are processed from larger primary transcripts and
range from 20-23 nucleotides in length, have emerged as a hot topic
in molecular biology research because of their important roles in a
wide range of biological processes, including gene regulation, cell
differentiation, growth, and development, as well as certain
disease states. Other small RNA molecules, such as siRNAs, are also
involved in gene silencing and genome modification.
[0003] The long delay to the realization of the existence and
importance of small RNA could, in part, be attributed to the fact
that small RNA molecules are often unintentionally eliminated
because of their small sizes from preparations of natural RNA
populations. Furthermore, small RNA molecules represent a very
small fraction in terms of weight of the total RNA population, and
without removal of abundant RNAs and enrichment of small RNAs,
their detection could be severely hampered. Historically,
variations of two methods have been used to isolate RNA from
biological samples. The first method relies on chemical extraction
with organic solvents such as phenol and chloroform under acidic
conditions to separate DNA and other biomolecules from the RNA,
which is then concentrated by alcohol precipitation. Alcohol
precipitation, however, does not quantitatively recover small RNA
molecules. The second method relies on immobilization of RNA on a
solid support binding matrix, such as silica. For this, the
RNA-containing sample is mixed with a high salt solution or a salt
and alcohol mixture to decrease the affinity of RNA for water and
increase its affinity for the silica matrix. Small RNA, however,
binds poorly to the support matrix under the conditions routinely
used. Thus, most existing RNA preparation methods and commercial
RNA purification kits are deficient in capturing small RNA.
[0004] With the recent surge of interest in miRNA and other small
RNA molecules, the standard isolation procedures have been modified
to facilitate the isolation of small RNA. These methods largely
rely on phenol and chloroform extraction and step-wise alcohol
fractionation. For example, U.S. Publication No. 2005/0059024
discloses a method in which a cell lysate is extracted with phenol
and chloroform to partition the genomic DNA into an interphase
between an organic lower phase and an aqueous upper phase. The
aqueous upper phase is collected and mixed with a low percentage of
alcohol and applied to a first binding matrix. The large RNA is
immobilized onto the first matrix and the small RNA flow through
the matrix. The flow-through fraction is then mixed with a higher
percentage of alcohol and applied to a second binding matrix, to
which the small RNA binds and can be recovered. Thus, small RNA can
be isolated and purified using a multi-step procedure. A major
drawback of the current methodology is the use of phenol and
chloroform, not only because they pose potential health hazards but
also because they are ineffective with certain biological material,
such as plant tissues that are rich in phenolic or polyphenolic
compounds. Another drawback of the current methodology is that
phase separation and alcohol fractionation are laborious and time
consuming, making them incompatible with high throughput and
automation demands.
[0005] The present invention provides methods and compositions for
the rapid isolation of small RNA from a variety of biological
sources without using phenol and chloroform extraction or alcohol
gradient fractionation.
SUMMARY OF THE INVENTION
[0006] The present invention encompasses compositions and methods
to rapidly and efficiently isolate small RNA molecules from a
biological sample. One aspect of the invention is an extraction
composition comprising a chaotropic agent and a metal salt.
[0007] Another aspect of the invention provides a method for
isolating small RNA from a biological sample. In the method, the
biological sample is contacted with a chaotropic agent and a metal
salt. After contact, small RNA is released from debris in the
biological sample. The small RNA remains in solution, allowing it
to be separated from the debris by a variety of methods known in
the art.
[0008] A further aspect of the invention encompasses a kit
comprising solutions to prepare an extraction composition, all
concomitant agents and buffers, means to isolate the small RNA, and
complete instructions.
[0009] Other aspects and features of the invention will be in part
apparent and in part described in more detail herein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0010] It has been discovered that contacting a biological sample
with a chaotropic agent and a metal salt leads to the release of
small RNA from other biomolecules. In particular, contact with the
chaotropic agent and metal salt selectively precipitates the large
RNA, genomic DNA, and other large macromolecules, whereas the small
RNA remains in solution. The small RNA may be readily separated and
isolated from the aggregated macromolecules. As illustrated in the
examples, the methods and compositions of the present invention
allow the rapid isolation of pure preparations of small RNA in high
yield from a variety of organisms, including, plant tissue,
mammalian cultured cells, mammalian tissue, yeast cells, and
bacterial cells.
I. Extraction Compositions
[0011] One aspect of the invention encompasses an extraction
composition. Typically the extraction composition will have a
chaotropic agent and a metal salt. In this context, the term
"composition" is used in its broadest sense to mean use of a
chaotropic agent and metal salt for the separation of small RNA
from a biological sample. The term composition does not mean that
the two agents have to be contacted with the biological sample at
the same time as a part of the same solution. It is contemplated
for example, as described below, that the chaotropic agent and
metal salt may be contacted with the biological sample either
simultaneously as part of the same mixture or added sequentially,
one reagent after the other. As will be appreciated by a skilled
artisan, the extraction composition may optionally include a
variety of other agents without departing from the scope of the
invention. Suitable non-limiting examples of agents comprising the
extraction composition are detailed below.
(a) Chaotropic Agent
[0012] A variety of chaotropic agents are suitable for use in the
extraction composition. Generally speaking, the chaotropic agent
denatures proteins, disrupts membranes, releases nucleic acids,
protects RNA from degradation, and facilitates cell lysis. Examples
of suitable chaotropic agents include guanidine hydrochloride,
guanidine thiocyanate, guanidine carbonate, sodium iodide, sodium
perchlorate, sodium trichloroacetate, urea, and thiourea. The
chaotropic agent may be incorporated into the extraction
composition alone or as a combination of two or more chaotropic
agents. As will be appreciated by one skilled in the art, the
choice of chaotropic agent will be determined by the origin of
material from which small RNA is to be isolated. In one embodiment,
the chaotropic agent is guanidine thiocyanate. Guanidine
thiocyanate, however, is not particularly suitable for RNA
isolation from certain plant tissues, such as cotton leaves, grape
leaves, red maple leaves, and gymnosperm conifer needles, which are
rich in phenolic or polyphenolic compounds. In another embodiment,
the chaotropic agent is a combination of two or more quanidinium
salts. In a preferred embodiment, the chaotropic agent is guanidine
hydrochloride.
[0013] The concentration of the chaotropic agent or the combination
of chaotropic agents in the extraction composition may and will
vary but may range from about 1 M to about 8 M. Lower
concentrations of a chaotropic agent may be used if cell disruption
and RNase inhibition are not major concerns. In one aspect, the
concentration of the chaotopic agent is about 3 M. In another
aspect, the concentration of the chaotopic agent is about 6 M. In
another aspect, the concentration of the chaotopic agent is about 4
M. In yet another aspect, the concentration of the chaotopic agent
is about 5 M.
(b) Metal Salt
[0014] The extraction composition includes at least one metal salt.
A variety of metal salts are suitable for use in the invention. The
metal salt may be incorporated into the extraction composition
before or after contacting the biological sample. The metal salt
may be a group IA metal salt or a group IIA metal salt. Suitable
examples of group IIA metals include beryllium, magnesium, calcium,
strontium, and barium. Suitable examples of group IA metals include
lithium, sodium, potassium cesium, and francium. In a preferred
embodiment, the metal salt is a lithium salt. Examples of suitable
lithium salts include lithium acetate, lithium borate, lithium
carbonate, lithium chloride, and lithium citrate. In a preferred
embodiment, the lithium salt is lithium chloride.
[0015] The concentration of metal salt or combination of metal
salts may range from about 1 M to about 8 M. In one aspect, the
concentration of lithium salt ranges from about 1.5 M to about 6 M.
In one embodiment, the concentration of lithium chloride is about 6
M. In another embodiment, the concentration of lithium chloride is
about 2.4 M. In another embodiment, the concentration of lithium
chloride is about 1.8 M. In yet another embodiment, the
concentration of lithium chloride is about 3.6 M.
[0016] Without being bound by any particular theory, it is believed
that the combination of a chaotropic agent and a lithium salt in
the extraction composition creates a discriminating environment
that is particularly suitable for the separation of large RNA from
small RNA. It is known that Li.sup.+ ions have a very high
charge/radius ratio and a unique affinity for RNA molecules. They
can effectively neutralize the negative charges on the RNA backbone
and remove much of the water shell from the RNA molecule. A
chaotrope, on the other hand, has a strong disrupting ability,
which can keep the charge-neutralized RNA molecules from collapsing
on each other and becoming aggregated. As a result of the
counteraction, each charge-neutralized RNA molecule may behave as a
discrete entity in the extraction composition. It is further
believed that charge-neutralized large RNAs possess a higher
density than the extraction composition and, therefore, they are
very susceptible to precipitation, whereas charge-neutralized small
RNAs have a lower density than the extraction composition and,
therefore, they substantially remain in solution. The density of
each RNA molecule may also be affected to some extent by pH, for
H.sup.+ can compete with Li.sup.+ for the negative charges on the
RNA backbone. As a consequence, the extraction composition is
optimized for extracting small RNA, as detailed below.
(3) pH and Buffer
[0017] It has been discovered, as detailed in the examples, that
the pH of the extraction composition differentially affects the
solubility of small RNA, large RNA, and genomic DNA. At values
below about pH 4, large RNA and genomic DNA are insoluble and
precipitate out of solution, whereas the small RNA is substantially
soluble and stays in solution. As pH values rise above about pH 4,
the small RNA remains soluble and the large RNA remains insoluble,
but the solubility of DNA increases.
[0018] In order to maintain a desired pH for optimizing small RNA
isolation, therefore, a buffer is typically incorporated into the
extraction composition. In one embodiment, the pH of the extraction
composition ranges from about 3 to about 8. In an alternative
embodiment, the extraction composition has a pH of about 7. In
another embodiment, the pH of the extraction composition is less
than about 5.0 and more preferably, is less than about 4.0. In an
alternative embodiment, the extraction composition has a pH that
ranges from about 3.0 to about 4.0. In yet another embodiment, the
extraction composition has a pH of about 3.5.
[0019] A variety of buffers are suitable for use in the extraction
composition. By way of non-limiting example, the buffers may
include, but are not limited to, trizma acetate, EDTA, tris,
glycine, and citrate. EDTA also has the ability to chelate
Mg.sup.2+ ions, thereby inactivating nucleases. In one aspect, the
buffer is EDTA. In a preferred aspect, the buffer is trizma
acetate. The buffer may be incorporated into the extraction
composition alone or as a combination of two or more buffers. The
concentration of buffer is typically sufficient to maintain a
desired pH range. In one embodiment, the concentration of buffer in
the extraction composition may range from about 20 mM to about 100
mM. In other embodiment, the concentration of the buffer in the
extraction composition may range from about 30 mM to about 50 mM.
In a further embodiment, the concentration of buffer in the
extraction composition is about 40 mM.
(4) Detergent
[0020] The extraction composition may optionally include one or
more detergents. A variety of detergents may be utilized in the
present invention. Generally speaking, the detergent will typically
promote protein solubilization, membrane disruption, and cell
permeabilization. Detergents are preferably included in certain
embodiments when small RNA is separated from certain plant tissues
that are rich in phenolic or polyphenolic compounds. Examples of
such plant tissues may include, but are not limited to, cotton
leaves, grape leaves, red maple leaves, and gymnosperm conifer
needles.
[0021] Examples of suitable detergents that may be incorporated
into the extraction composition are polyoxyethylene detergents and
quaternary ammonium compounds. Polyoxyethylene detergents are
nonionic, while quaternary ammonium compounds are cationic.
Non-limiting examples of polyoxyethylene detergents include
polyoxyethylenesorbitan monolaurate (Tween 20, Sigma-Aldrich, St.
Louis, Mo.), polyoxyethylenesorbitan monooleate (Tween 80,
Sigma-Aldrich, St. Louis, Mo.), octylphenoxy poly(ethyleneoxy)
ethanol (Igepal CA 630, Sigma-Aldrich, St. Louis, Mo.), and
t-octylphenoxypolyethoxyethanol (Triton X100 and Triton X114,
Sigma-Aldrich, St. Louis, Mo.), and P-40 (NP-40, Sigma-Aldrich, St.
Louis, Mo.). Non-limiting examples of quaternary ammonium compounds
include hexadecyltrimethylammonium bromide (CTAB, Sigma-Aldrich,
St. Louis, Mo.), dodecyltrimethylammonium bromide,
ethylhexadecyidimethylammonium bromide, benzethonium chloride
(Hyamine 1622, Sigma-Aldrich, St. Louis, Mo.), and
benzyldimethylhexadecylammonium chloride. The detergents may be
incorporated in the extraction composition alone or as a
combination of two or more detergents. In one embodiment, the
detergent is Triton X100. In another embodiment, the detergent is
Igepal. In a preferred embodiment, the detergent is Tween 20.
[0022] As will be appreciated by a skilled artisan, the
concentration of detergent present in the extraction composition
can and will vary. In one embodiment, the detergent concentration
is between about 0.1% to about 10% by weight. In another
embodiment, the detergent concentration is between about 1% and
about 5% by weight. In still another embodiment, the detergent
concentration is between about 1% and about 2% by weight.
(5) Thiol-Reducing Agent
[0023] The extraction composition may also comprise a
thiol-reducing agent to block the formation of disulfide bonds upon
cell disruption and protein denaturation, thereby keeping
endogenous RNases inactive. Suitable thiol-reducing agents include
dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethylamine, and
tris(carboxyethyl) phosphine (TCEP). In one embodiment, the
thiol-reducing agent is DTT, with a concentration between about 1
mM and about 10 mM. In another aspect, the thiol-reducing agent is
2-mercaptoethanol. In one embodiment, the concentration of
2-mercaptoethanol is between about 0.1% to about 2% by weight. In
yet another aspect, the concentration of 2-mercaptoethanol is about
1% by weight.
(6) Antifoaming Agent
[0024] Depending upon the source of the biological sample, an
antifoaming agent may optionally be incorporated into the
extraction composition. Antifoaming agents may be an organic
antifoaming agent or a silicone-based antifoaming agent. Examples
of organic antifoaming agents include Antifoam 204 and Antifoam
O-30. Examples of silicone-based antifoaming agents include
Antifoam A, Antifoam B, Antifoam C, Antifoam Y-30, and Sag 471. The
concentration of antifoam agent is typically sufficient to ensure
adequate defoaming. The concentration of an organic antifoam agent
may be within the range from 0.005% to 0.01% by weight. The
concentration of a silicone-based agent may be within the range
from 1 ppm to 100 ppm.
(7) Bulking Agent
[0025] A bulking agent may optionally be incorporated into the
extraction composition to facilitate the precipitation of nucleic
acids. Bulking agents typically selectively promote the
precipitation of large nucleic acids compared to small nucleic
acids. In one embodiment, a bulking agent may be added to the
extraction composition to promote the precipitation of large RNA
and genomic DNA. In another embodiment, a bulking agent may be
added to the extraction composition to discriminate between the
different sized molecules of small RNA.
[0026] Several bulking agents are suitable for use in the present
invention. A bulking agent may be nonionic or ionic. Nonionic
bulking agents include alcohols and hydrophilic neutral polymers.
Exemplary alcohols that may be used as nonionic bulking agents
include butanol, ethanol, isopropanol, methanol, and propanol.
Hydrophilic neutral polymers that may be used as nonionic bulking
agents include dextran sulfate, polyethylene glycol (PEG),
tetraethylene glycol, and polyvinylpyrrolidine (PVP). The
concentration of a nonionic bulking agent or the combination of
nonionic agents may range from about 3% to about 10% by weight.
Ionic bulking agents include cationic detergents and polyamines.
Examples of ionic bulking agents include hexadecyltrimethylammonium
bromide (CTAB), dodecyltrimethylammonium bromide, spermine, and
spermidine. Other polyamines, or their derivatives, and other
cationic detergents also may be used as ionic bulking agents. The
concentration of an ionic bulking agent or the combination of ionic
agents may range from about 10 mM to about 100 mM, but other
concentrations also may be useful. In one aspect, the bulking agent
is the nonionic agent, isopropanol. In another aspect, the nonionic
bulking agent ethanol is incorporated into the extraction
composition. In yet another aspect, the ionic bulking agent
spermidine is incorporated into the extraction composition.
[0027] The extraction compositions of the invention include any
combination of chaoptropic agents and metal salts detailed herein.
The extraction composition may optionally include, in addition to
the chaoptropic agent and metal salt, any of the buffers,
detergents, thiol-reducing agents, antifoaming agents, bulking
agents detailed herein or otherwise known in the art to be useful
to isolate small RNA from a biological sample. Non-limiting
examples of extraction compositions of the invention are detailed
in Table A. Suitable examples of extraction compositions of the
invention detailed in Table A include the listed chaotropic agent
and a metal salt and optionally include any of the agents listed as
"other agents". TABLE-US-00001 TABLE A Chaotropic agent Metal salt
Other agents guanidine hydrochloride lithium chloride buffer,
detergent, thiol- reducing agent, antifoaming agent, bulking agent
guanidine hydrochloride lithium acetate buffer, detergent, thiol-
reducing agent, antifoaming agent, bulking agent guanidine
hydrochloride lithium borate buffer, detergent, thiol- reducing
agent, antifoaming agent, bulking agent guanidine hydrochloride
lithium carbonate buffer, detergent, thiol- reducing agent,
antifoaming agent, bulking agent guanidine hydrochloride lithium
citrate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent guanidine thiocyanate lithium chloride buffer,
detergent, thiol- reducing agent, antifoaming agent, bulking agent
guanidine thiocyanate lithium acetate buffer, detergent, thiol-
reducing agent, antifoaming agent, bulking agent guanidine
thiocyanate lithium borate buffer, detergent, thiol- reducing
agent, antifoaming agent, bulking agent guanidine thiocyanate
lithium carbonate buffer, detergent, thiol- reducing agent,
antifoaming agent, bulking agent guanidine thiocyanate lithium
citrate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent guanidine carbonate lithium chloride buffer,
detergent, thiol- reducing agent, antifoaming agent, bulking agent
guanidine carbonate lithium acetate buffer, detergent, thiol-
reducing agent, antifoaming agent, bulking agent guanidine
carbonate lithium borate buffer, detergent, thiol- reducing agent,
antifoaming agent, bulking agent guanidine carbonate lithium
carbonate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent guanidine carbonate lithium citrate buffer,
detergent, thiol- reducing agent, antifoaming agent, bulking agent
sodium iodide lithium chloride buffer, detergent, thiol- reducing
agent, antifoaming agent, bulking agent sodium iodide lithium
acetate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent sodium iodide lithium borate buffer,
detergent, thiol- reducing agent, antifoaming agent, bulking agent
sodium iodide lithium carbonate buffer, detergent, thiol- reducing
agent, antifoaming agent, bulking agent sodium iodide lithium
citrate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent sodium perchlorate lithium chloride buffer,
detergent, thiol- reducing agent, antifoaming agent, bulking agent
sodium perchlorate lithium acetate buffer, detergent, thiol-
reducing agent, antifoaming agent, bulking agent sodium perchlorate
lithium borate buffer, detergent, thiol- reducing agent,
antifoaming agent, bulking agent sodium perchlorate lithium
carbonate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent sodium perchlorate lithium citrate buffer,
detergent, thiol- reducing agent, antifoaming agent, bulking agent
sodium trichloroacetate lithium chloride buffer, detergent, thiol-
reducing agent, antifoaming agent, bulking agent sodium
trichloroacetate lithium acetate buffer, detergent, thiol- reducing
agent, antifoaming agent, bulking agent sodium trichloroacetate
lithium borate buffer, detergent, thiol- reducing agent,
antifoaming agent, bulking agent sodium trichloroacetate lithium
carbonate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent sodium trichloroacetate lithium citrate
buffer, detergent, thiol- reducing agent, antifoaming agent,
bulking agent urea lithium chloride buffer, detergent, thiol-
reducing agent, antifoaming agent, bulking agent urea lithium
acetate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent urea lithium borate buffer, detergent, thiol-
reducing agent, antifoaming agent, bulking agent urea lithium
carbonate buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent urea lithium citrate buffer, detergent, thiol-
reducing agent, antifoaming agent, bulking agent thiourea lithium
chloride buffer, detergent, thiol- reducing agent, antifoaming
agent, bulking agent thiourea lithium acetate buffer, detergent,
thiol- reducing agent, antifoaming agent, bulking agent thiourea
lithium borate buffer, detergent, thiol- reducing agent,
antifoaming agent, bulking agent thiourea lithium carbonate buffer,
detergent, thiol- reducing agent, antifoaming agent, bulking agent
thiourea lithium citrate buffer, detergent, thiol- reducing agent,
antifoaming agent, bulking agent
II. Methods for Isolating Small RNA
[0028] The extraction compositions of the present invention may be
utilized to isolate small RNA molecules from a biological sample.
Typically, small RNA molecules are less than about 200 nucleotides
in length. Both prokaryotic and eukaryotic cells contain a
plurality of different sized RNA molecules. RNA molecules with
lengths greater than about 200 nucleotides include messenger RNA
(mRNA), 16S/18S ribosomal RNA (rRNA), and 23S/28S rRNA. Small RNA
molecules with lengths less than about 200 nucleotides include 5.8S
rRNA, 5S rRNA, transfer RNA (tRNA), small nuclear RNA (snRNA),
small nucleolar RNA (snoRNA); micro RNA (miRNA), small interfering
RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA
(rasiRNA), small temporal RNA (stRNA), tiny non-coding RNA
(tncRNA), small scan RNA (scRNA), and small modulatory RNA
(smRNA).
[0029] In the isolation method of the invention, the biological
sample is contacted with any of the extraction compositions
disclosed herein. Generally speaking, the extraction composition
will comprise a chaotropic agent and a metal salt. The biological
sample may be contacted with the chaotropic agent and the metal
salt simultaneously. Alternatively, the biological sample may
contacted with the chaotropic agent and the metal salt
sequentially, one reagent after the other. Contact with the
extraction composition releases the small RNA from the debris
present in the biological sample, such as the large biomolecules,
which become insoluble and precipitate out of solution. The
precipitated molecules include large RNA, genomic DNA, and other
macromolecules, (i.e., collectively referred to as "debris"). The
small RNA remains substantially soluble in the extraction
composition.
[0030] Small RNA may be isolated from a variety of biological
samples. Examples of a suitable biological sample include a cell, a
tissue from a multicellular organism, a whole organism, a virus, a
body fluid, such as serum, blood, saliva, urine, or cerebrospinal
fluid, or any other nucleic acid-containing preparation.
[0031] As will be appreciated by a skilled artisan, the biological
sample may be contacted with the extraction composition by several
suitable methods generally known in the art. In one embodiment,
cells are lysed upon contact with the extraction composition. In
another embodiment, tissue is ground to a fine powder in liquid
nitrogen and then mixed with the extraction composition. In another
embodiment, tissue is homogenized in the extraction composition in
a rotor-stator homogenizer, a pestle-type homogenizer, or a
blender. In yet another embodiment, fungal or bacterial cells are
chemically treated with enzymes or physically pulverized with beads
to disrupt the cell wall prior to being contacted with the
extraction composition. In a further embodiment, a nucleic
acid-containing preparation is contacted with the extraction
composition. In general, contact with the extraction composition
causes the selective denaturation and aggregation of the large
biomolecules and the formation of debris in the mixture. The small
RNA, however, remains in solution and may be separated from the
debris and purified from the mixture.
[0032] Separation of the small RNA may be accomplished by several
methods well known in the art. In one embodiment, the aggregated
debris is separated from the small RNA-containing mixture by
centrifugation. In another embodiment, the aggregated debris is
separated from the small RNA-containing mixture by filtration. In
another embodiment, the small RNAs are separated from the debris by
chromatography. In an exemplary embodiment, the debris is removed
by centrifugation and filtration, and the small RNA is isolated
from the soluble mixture by chromatography.
[0033] Suitable examples of chromatographic methods include size
exclusion chromatography and affinity chromatography. In a
preferred embodiment, the small RNAs are isolated by affinity
chromatography. Examples of suitable affinity binding matrices
include any solid matrix, as well as any coated surface to which
nucleic acids bind. In one embodiment, the binding matrix is a
hydrophilic matrix. The hydrophilic matrix may be an organic
binding matrix or an inorganic binding matrix. Examples of suitable
organic hydrophilic matrices include, but are not limited to,
acrylic copolymers, cellulose, dextran, agarose, and acrylic amide.
Suitable examples of inorganic hydrophilic matrices include, but
are not limited to, silica, borosilicate, diatomaceous earth,
aluminum oxides, glass, titanium oxides, zirconium oxides, and
hydroxyapatite. In one embodiment, the binding matrix is a
silica-based binding matrix. Examples of silica matrices include,
but are not limited to, silica particles, silica filters, and
magnetized silica. In a preferred embodiment, the binding matrix is
a filter comprising borosilicate fibers.
[0034] Small RNA typically binds to silica-based binding matrices
in the presence of a chaotropic salt and a high concentration of
alcohol. Alcohols that may be added to the small RNA-containing
mixture, to facilitate the binding of small RNA to the binding
matrix, include ethanol, isopropanol, butanol, methanol, and
propanol. The alcohols may be used alone or in combination of two
or more alcohols. In one embodiment, the alcohol added to the
binding mixture is ethanol. In other embodiment, the alcohol added
to the binding mixture is isopropanol. The concentration of the
alcohol or combination of two or more alcohols in the binding
mixture is preferentially greater than about 50%. In one aspect,
the concentration of ethanol in the binding mixture is about 67%.
In another aspect, the concentration of ethanol in the binding
mixture is about 55%. Upon binding of the small RNA to the silica
or borosilicate binding matrix, impurities are removed with high
salt wash solutions and alcohol wash solutions. Examples of high
salt wash solutions include, but are not limited to, 12 M LiCl and
9 M LiCl. Examples of alcohol wash solutions include, but are not
limited to, 100% ethanol and 80% ethanol. Small RNAs are eluted
from the binding matrix with RNase-free water or an RNase-free low
salt buffer.
III. Kits for Isolating Small RNA
[0035] The extraction composition and the method of the present
invention may be combined to create a kit for the isolation of
small RNA. In one embodiment, the kit comprises solutions to
prepare an extraction composition of the invention and instructions
for use. In a preferred embodiment, the kit comprises solutions to
prepare an extraction composition of the invention, concomitant
additive agents, a separation means, companion wash and elution
solutions, and complete instructions for isolating the small RNA.
In an exemplary embodiment, the separation means provided in the
complete kit is a binding filter comprising borosilicate
fibers.
Definitions
[0036] The term "biological sample" as used herein refers to any
nucleic acid-containing material derived from any source, either in
vivo or in vitro. The biological sample may be a eukaryotic or a
prokaryotic cell, a tissue from a multicellular organism, a whole
organism, a virus, a body fluid, such as serum, blood, saliva,
urine, semen, or cerebrospinal fluid, or a mixture of nucleic acids
generated in vitro.
[0037] The terms "biomolecules" or "macromolecules" used herein
refer to large RNA, DNA, proteins, carbohydrates, lipids, and
combinations thereof.
[0038] The term "bulking agent" used herein refers to a compound
that effectively increases the concentration of nucleic acids
because the nucleic acids are excluded from the space occupied by
the bulking agent.
[0039] The term "chaotropic agent" refers to an agent that disrupts
the secondary or higher structure of certain molecules, such that
the molecule unfolds and loses biological activity.
[0040] The term "debris" used herein refers to the insoluble RNA,
DNA, and other biomolecules that precipitate or aggregate upon
contact with the extraction composition.
[0041] The term "extraction" refers to the release from or the
separation of a specific molecule from a mixture of molecules. More
specifically, it refers to the process by which small RNA is
released from other biomolecules upon contact with the extraction
composition, due to the precipitation of the biomolecules upon
contact with the extraction composition.
[0042] The term "immobilization" refers to adherence or binding of
the target molecule (i.e., small RNA) to a binding matrix.
[0043] The terms "isolate", "purify", or "separate" refer to the
removal of at least a portion of the small RNA from at least part
of the debris in a biological sample.
[0044] The term "lyse" or "lysis" refers to the rupturing of the
cell wall and/or cell membrane of a cell so that cellular contents
are released.
[0045] The term "small RNA" used herein refers to RNA molecules
with lengths of less than about 200 nucleotides. Small RNA
molecules may be single stranded or double stranded. Examples of
small RNA include, but are not limited to, 5.8 S rRNA, 5 S rRNA,
transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA
(snoRNA); micro RNA (miRNA), small interfering RNA (siRNA),
transacting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA),
small temporal RNA (stRNA), tiny non-coding RNA (tncRNA), small
scan RNA (scRNA), and small modulatory RNA (smRNA).
[0046] As various changes could be made in the above compounds,
products and methods without departing from the scope of the
invention, it is intended that all matter contained in the above
description and in the examples given below, shall be interpreted
as illustrative and not in a limiting sense.
EXAMPLES
[0047] The following examples illustrate the invention.
Example 1
Effects of pH on Nucleic Acid Separation
[0048] Nine basal solutions were prepared that each comprised 7 M
guanidine hydrochloride, 60 mM trizma acetate, and 2% Tween 20, but
each had a different pH through titration with acetic acid or NaOH.
The pH values were 3.2, 3.4, 3.6, 3.8, 4.0, 5.0, 6.0, 7.0, and 8.0.
Nine lysis solutions were prepared by combining each of the nine
basal solutions with a 12 M LiCl solution in a 7:3 ratio. The
resulting lysis solutions each comprised 4.9 M guanidine
hydrochloride, 3.6 M LiCl, 42 mM trizma acetate, and 1.4% Tween 20,
and each solution had a different pH. Each lysis solution was
further supplemented with 2-mercaptoethanol at 1%.
[0049] Grape leaves were ground to a fine powder in liquid nitrogen
and nine 100-mg aliquots were prepared from the powdered material.
Each aliquot was lysed in 750 .mu.l of a lysis solution at
55.degree. C. for 4 minutes. The samples were then centrifuged for
5 minutes performed. The supernatant fraction was filtered through
a filtration column (C 6866, Sigma-Aldrich, St. Louis, Mo.) by 1
minute of centrifugation to remove carry-over particulates. The
clarified lysate was mixed with 830 .mu.l of 100% ethanol and
applied to a silica filter binding column (C6991, Sigma-Aldrich,
St. Louis, Mo.) in two loadings, with 30 seconds of centrifugation
after each loading. The column washed in succession with 500 .mu.l
of 100% ethanol, 500 .mu.l of 12 M LiCl, and twice with 500 .mu.l
of an alcohol wash solution comprising 80% ethanol and 10 mM tris
(pH 7.0). Each wash step was carried out with a short
centrifugation (30 seconds or 1 minute). The column was dried by 1
minute of centrifugation and the bound nucleic acids were eluted in
50 .mu.l of RNase-free water and 1 minute of centrifugation. All
centrifugation steps were performed in a bench-top microcentrifuge
at top speed (14,000.times.g) at room temperature. The samples were
analyzed by reading the UV absorbance in a spectrophotometer and
electrophoresing 0.5 .mu.g of each sample on a 2% agarose gel.
[0050] The amount of RNA recovered under each lysis condition is
presented in Table 1. The A.sub.260/280 ratios were between 2.1 and
2.2 for each sample. Following agarose gel electrophoresis, no
bands of 18S and 25S rRNA were detected in any of the samples. A
strong band of small RNA with a mobility similar to a yeast tRNA
standard (70-80 nucleotides) was detected in all samples. Some
minor bands of small RNA with mobilities slightly slower than the
strong band of small RNA were detectable in the samples prepared
with the lysis solutions at pH below 3.8. A genomic DNA band with a
mobility slower than a 10 kb DNA marker was detected in the samples
prepared with the lysis solutions at pH above 4. The intensity of
this genomic DNA band increased as the lysis solutions became more
basic. The results indicated that large RNA was in an insoluble
form in the lysis solutions regardless of pH and was removed from
all preparations, and that samples prepared with the lysis
solutions at pH 4 or lower consisted primarily of small RNA and
were substantially free of genomic DNA. TABLE-US-00002 TABLE 1
Effect of pH on Yield. Condition Yield pH 3.2 12.8 .mu.g pH 3.4
11.8 .mu.g pH 3.6 11.9 .mu.g pH 3.8 12.0 .mu.g pH 4.0 9.2 .mu.g pH
5.0 7.4 .mu.g pH 6.0 7.4 .mu.g pH 7.0 7.9 .mu.g pH 8.0 8.7
.mu.g
Example 2
Effects of a Nonionic Bulking Agent
[0051] A basal solution was prepared comprising 7 M guanidine
hydrochloride, 2% Tween 20, and 60 mM trizma acetate, pH 3.4. The
basal solution was then combined with a 12 M LiCl solution and
ethanol in some formulations in different ratios to form 6 lysis
solutions, as detailed in Table 2. Each lysis solution was further
supplemented with 2-mercaptoethanol at 1%. TABLE-US-00003 TABLE 2
Composition of Lysis Solutions. Basal Solution # Solution LiCl
Solution Ethanol 1 80% 20% -- 2 74% 20% 6% 3 70% 20% 10% 4 70% 30%
-- 5 64% 30% 6% 6 60% 30% 10%
[0052] Grape leaf samples (100 mg each) were prepared as described
above. Each sample was lysed in 750 .mu.l of a lysis solution at
55.degree. C. for 4 minutes. Small RNA was purified as described in
Example 1. The samples were analyzed by reading the UV absorbance
in a spectrophotometer and running 0.5 .mu.g of each sample on a 4%
agarose gel.
[0053] The amount of RNA recovered under each lysis condition is
presented in Table 3. The A.sub.260/280 ratios were between 2.1 and
2.2 for each sample. Following agarose gel electrophoresis, no
bands of 18S and 25S rRNA or genomic DNA were detected in any of
the samples. A very strong band of RNA with a mobility similar to a
tRNA standard (70-80 nucleotides) was present in all samples. In
addition, two minor bands of small RNA with mobilities slightly
slower than the major band of small RNA were detected in the
samples that were prepared with Lysis Solutions #1 and #4, which
did not contain ethanol as an additive. These two minor bands of
small RNA are most likely 5S rRNA (about 120 nucleotides) and 5.8S
rRNA (about 150 nucleotides). The intensity of these minor bands
were greatly reduced in the samples that were prepared with Lysis
Solutions #2 and #5, which contained 6% ethanol as an additive, and
they were further reduced to nearly undetectable levels in the
samples prepared with Lysis Solution #3 and #6, which contained 10%
ethanol as an additive. The results demonstrated that ethanol may
be used as a nonionic additive in the process of the present
invention to discriminate among different sized molecules of small
RNA. TABLE-US-00004 TABLE 3 Effects of Ethanol on Yield. Sample #
Yield 1 12.2 .mu.g 2 8.5 .mu.g 3 5.9 .mu.g 4 11.6 .mu.g 5 9.3 .mu.g
6 6.1 .mu.g
Example 3
Effects of an Ionic Bulking Agent
[0054] A lysis solution was prepared comprising 7.2 M guanidine
hydrochloride, 2% Tween 20, and 50 mM trizma acetate, pH of 7.0.
The lysis solution was further supplemented with 2-mercaptoethanol
at 1%. A mouse liver tissue sample (30 mg) was homogenized in 300
.mu.l of the lysis solution with a rotor-stator homogenizer.
Following homogenization, 3 .mu.l of 1 M spermidine solution in
water was added into the lysate. The mixture was incubated on ice
for 5 minutes and centrifuged for 5 minutes to precipitate the
genomic DNA. The supernatant was collected and mixed with 1 volume
of a 12 M LiCl solution. The sample was centrifuged for 5 minutes
to precipitate the large RNA. The supernatant was filtered through
a filtration column (C 6866, Sigma-Aldrich, St. Louis, Mo.) with 30
seconds of centrifugation to remove carry-over particulates.
[0055] The flow-through was mixed with 1.25 volumes of 100% ethanol
and the mixture was applied to a silica filter binding column
(C6991, Sigma-Aldrich, St. Louis, Mo.). The column washed once with
300 .mu.l of 12 M LiCl and twice with 500 .mu.l of an alcohol wash
solution comprising 80% ethanol and 10 mM tris (pH 7.0). The column
was dried and bound nucleic acids were eluted in 50 .mu.l of
RNase-free water. The binding, washing, column drying, and eluting
steps were assisted by a short centrifugation (30 seconds or 1
minute). All centrifugation steps were carried out at top speed
(14,000.times.g) at room temperature. The sample was analyzed by
reading the UV absorbance in a spectrophotometer and resolving 0.5
.mu.g of each sample on a 4% agarose gel.
[0056] The yield was 4.5 .mu.g, and the A.sub.260/280 ratio was
2.1. Only a single band of small RNA with a mobility similar to a
tRNA standard (70-80 nucleotides) was detected on the agarose gel.
No genomic DNA or large RNA bands were detectable. The results
demonstrate that spermidine may be used as an ionic additive to
remove genomic DNA when biological samples are lysed under high pH
conditions.
Example 4
Purification from Mammalian Culture Cells
a) Hela Cell Adherent Culture
[0057] Hela cells were cultured in a T125 flask in DMEM medium with
10% FBS to near 100% confluence. Cells were detached from the flask
with a trypsin/EDTA solution and then diluted in culture medium.
Aliquots, each containing about 3 million cells, were prepared and
the medium was subsequently removed by centrifugation. The cell
pellet samples were flash-frozen in liquid nitrogen and stored at
-70.degree. C. before use. One of the frozen cell samples was lysed
for 5 minutes at room temperature in 500 .mu.l of a lysis solution
comprising 3 M guanidine hydrochloride, 6 M LiCl, 25 mM EDTA, 0.75%
Tween 20, and 1% 2-mercaptoethanol, pH 3.5. The lysis solution was
prepared by combining 0.5 volumes of a basal solution (6 M
guanidine hydrochloride, 50 mM EDTA, 1.5% Tween 20, pH 3.5) with
0.5 volumes of a 12 M LiCl solution and 0.01 volume of
2-mercaptoethanol. The sample was then centrifuged for 6 minutes to
precipitate large RNA and genomic DNA. The supernatant was filtered
through a filtration column (C6866, Sigma-Aldrich, St. Louis, Mo.)
to remove carry-over particulates. Two volumes of 100% ethanol were
mixed with the flow-through and the mixture was applied to a silica
filter binding column (C6991, Sigma-Aldrich, St. Louis, Mo.). The
column was then washed twice with 500 .mu.l of an alcohol wash
solution comprising 80% ethanol and 10 mM tris at pH 7.0, and
subsequently dried. Bound nucleic acids were eluted in 50 .mu.l of
RNase-free water. The binding, washing, column drying, and eluting
steps were assisted by a brief centrifugation (30 seconds or 1
minute) at top speed in a microcentrifuge at room temperature.
Total RNA was prepared from the other frozen cell sample with a
total RNA purification kit (STRN50, Sigma-Aldrich, St. Louis, Mo.).
The samples were analyzed by reading the UV absorbance in a
spectrophotometer and running 0.5 .mu.g of each sample on a 2%
agarose gel.
[0058] The yields were 5 .mu.g for the preparation of selectively
isolated small RNAs and 58 .mu.g for the preparation of total RNA.
The A.sub.260/280 ratio was 2.1 for both samples. Only a prominent
band of small RNA running in front of the bromophenol blue tracking
dye was detected in the preparation of small RNA on the 2% agarose
gel. Two prominent bands of large RNA with mobilities much slower
than a 0.5 kb DNA marker were detected in the preparation of total
RNA.
b) HEK293 Cell Adherent Culture
[0059] HEK293 cells were cultured in a T25 flask in DMEM medium
with 10% FBS to near 100% confluence (about 4 million cells). The
culture medium was removed by aspiration and the culture washed
with 5 ml Hank's Balanced Salt Solution. Following the removal of
the wash solution, the culture was lysed for 5 minutes at room
temperature in 750 .mu.l of a lysis solution comprising 4.9 M
guanidine hydrochloride, 3.6 M LiCl, 42 mM trizma acetate, 1.4%
Tween 20, and 1% 2-mercaptoethanol, pH 3.4. The lysis solution was
prepared by combining 0.7 volumes of a basal solution (7 M
guanidine hydrochloride, 60 mM trizma acetate, 2% Tween 20, pH 3.4)
with 0.3 volumes of a 12 M LiCl solution and 0.01 volume of
2-mercaptoethanol. The lysate was then transferred to a 2-ml tube
and centrifuged for 5 minutes to precipitate the large RNA and
genomic DNA. The supernatant was filtered through a filtration
column (C 6866, Sigma-Aldrich, St. Louis, Mo.) and the flow-through
was mixed with 850 .mu.l of 100% ethanol. The mixture was applied
to a silica filter binding column (C6991, Sigma-Aldrich, St. Louis,
Mo.). The column was washed once with 500 .mu.l of 12 M LiCl and
twice with 500 .mu.l of an alcohol wash solution comprising 80%
ethanol and 10 mM tris, pH 7.0, and subsequently dried. Bound
nucleic acids were eluted in 50 .mu.l of RNase-free water. The
binding, washing, column drying, and eluting steps were each
assisted by a brief centrifugation (30 seconds or 1 minute) at top
speed in a microcentrifuge at room temperature. The sample was
analyzed by reading the UV absorbance in a spectrophotometer and
resolving 0.5 .mu.g of the sample on a 4% agarose gel.
[0060] The yield was 8.4 .mu.g, and the A.sub.260/280 ratio was
2.0. A prominent band of small RNA with a mobility similar to a
tRNA standard (70-80 nucleotides) and a few minor bands of small
RNA with mobilities slightly slower than the major band of small
RNA were detected. No bands of large RNA or genomic DNA were
detected.
c) K562 Suspension Culture Cells
[0061] K562 cells were grown in suspension in DMEM medium to late
stage. An aliquot of 2 million cells of the suspension culture was
centrifuged for 4 minutes and the medium was removed. The cell
pellet was lysed in 750 .mu.l of a lysis solution comprising 4.9 M
guanidine hydrochloride, 3.6 M LiCl, 42 mM trizma acetate, 1.4%
Tween 20, and 1% 2-mercaptoethanol, pH 3.4. Small RNA was purified
as described in the example of HEK293 adherence culture cells. The
sample was analyzed by reading the UV absorbance in a
spectrophotometer and running 0.5 .mu.g of the sample on a 4%
agarose gel.
[0062] The yield was 3 .mu.g, and the A.sub.260/280 ratio was 2.1.
A prominent band of small RNA with a mobility similar to a tRNA
standard (70-80 nucleotides) and a few minor bands of small RNAs
with mobilities slightly slower than the major band of small RNA
were detected on the 4% agarose gel. No bands of large RNA or
genomic DNA were detected.
Example 5
Purification from Mammalian Tissue
[0063] Mouse liver tissue (about 40 mg) was homogenized for about
30 seconds with a rotor-stator homogenizer in 750 .mu.l of a lysis
solution comprising 4.6 M guanidine hydrochloride, 3.6 M LiCl, 39
mM trizma acetate, 1.3% Tween 20, 5% ethanol, and 1%
2-mercaptoethanol, pH 3.4. The lysis solution was prepared by
combining 0.65 volumes of a basal solution (7 M guanidine
hydrochloride, 60 mM trizma acetate, 2% Tween 20, pH 3.4) with 0.3
volumes of a 12 M LiCl, 0.05 volumes of ethanol, and 0.01 volume of
2-mercaptoethanol. The homogenate was incubated at room temperature
for 5 minutes and centrifuged for 5 minutes at top speed at room
temperature. The supernatant was filtered through a filtration
column (C 6866, Sigma-Aldrich, St. Louis, Mo.) and the flow-through
was mixed with 970 .mu.l of 100% ethanol. Small RNA was then
purified by the silica column procedure as described in the example
of the HEK293 adherence culture cells. The sample was analyzed by
reading the UV absorbance in a spectrophotometer and
electrophoresing 0.5 .mu.g of the sample on a 4% agarose gel.
[0064] The yield was 19.2 .mu.g, and the A.sub.260/280 ratio was
2.0. Only a prominent band of small RNA with a mobility similar to
a tRNA standard (70-80 nucleotides) was detected on the 4% agarose
gel. No bands of large RNA or genomic DNA were detected.
Example 6
Purification from Yeast
[0065] Yeast (S. cerevisiae) cells were cultured in YPD medium
overnight. The OD.sub.600 of the culture was 1.54. An aliquot of
the culture containing approximately 4.6.times.10.sup.7 cells was
centrifuged at 12,000.times.g for 5 minutes and the culture medium
was removed. The cell pellet was resuspended in 25 .mu.l of Working
Yeast Digestion Solution (prepared freshly from Y0253 and Y0378 in
9 to 1 ratio, Sigma-Aldrich, St. Louis, Mo.). The sample was
incubated at room temperature for 10 minutes to digest the cell
wall. Following the digestion, the sample was lysed for 5 minutes
at room temperature in 750 .mu.l of a lysis solution comprising 5.6
M guanidine hydrochloride, 2.4 M LiCl, 48 mM trizma acetate, 1.6%
Tween 20, and 1% 2-mercaptoethanol, pH 3.4. The lysis solution was
prepared by combining 0.8 volumes of a basal solution (7 M
guanidine hydrochloride, 60 mM trizma acetate, 2% Tween 20, pH 3.4)
with 0.2 volumes of a 12 M LiCl solution and 0.01 volume of
2-mercaptoethanol. The lysate was centrifuged to precipitate the
large RNA and genomic DNA and the supernatant was filtered through
a filtration column as previously described. The clarified lysate
was mixed with 850 .mu.l of 100% ethanol before RNA binding. Small
RNA was then purified by the silica column procedure as described
in the example of the HEK293 adherence cells. The sample was
analyzed by reading the UV absorbance in a spectrophotometer and
running 0.25 .mu.g of the sample on a 4% agarose gel.
[0066] The yield was 5.3 .mu.g, and the A.sub.260/280 ratio was
2.1. A prominent band of small RNA with a mobility similar to a
tRNA standard (70-80 nucleotides) and two less prominent bands
(most likely the 5S rRNA and 5.8S rRNA) with mobilities slightly
slower than the major band of small RNA were detected on the 4%
agarose gel. No bands of large RNA or genomic DNA were
detected.
Example 7
Purification from Gram-Positive and Gram-Negative Bacteria
[0067] Bacillus subtilis (gram-positive) cells and E. coli
(gram-negative) cells were cultured in LB medium overnight. The
OD.sub.600 of the cultures was 4.4 and 4.0 for Bacillus subtilis
and E. coli, respectively. Aliquots of the cultures were prepared,
each containing approximately 1.times.10.sup.9 cells, and
centrifuged at 12,000.times.g for 5 minutes. Following removal of
the culture medium, a Bacillus and an E. coli cell pellet were each
resuspended in 25 .mu.l of Working Bacterial Digestion Solution
(prepared freshly from B7934 and B7809 in 9 to 1 ratio,
Sigma-Aldrich, St. Louis, Mo.). The samples were incubated at room
temperature for 10 minutes to digest the cell wall. Following the
digestion, each sample was lysed for 5 minutes at room temperature
in 750 .mu.l of a lysis solution comprising 5.95 M guanidine
hydrochloride, 1.8 M LiCl, 51 mM trizma acetate, 1.7% Tween 20, and
1% 2-mercaptoethanol, pH 3.4. The lysis solution was prepared by
combining 0.85 volumes of a basal solution (7 M guanidine
hydrochloride, 60 mM trizma acetate, 2% Tween 20, pH 3.4) with 0.15
volumes of a 12 M LiCl solution and 0.01 volume of
2-mercaptoethanol. An E. coli cell pellet sample was also lysed
with the lysis solution without prior enzyme digestion. Small RNA
was then purified as described in Example 6. The samples were
analyzed by reading UV absorbance in a spectrophotometer and
running 0.5 .mu.g of each sample on a 4% agarose gel.
[0068] The yields were 2.6 .mu.g, 4.5 .mu.g, and 3.7 .mu.g for
Bacillus subtilis culture, E. coli culture with enzyme digestion,
and E. coli culture without enzyme digestion, respectively. The
A.sub.260/280 ratio was 2.1 in all samples. A prominent band of
small RNA with a mobility similar to a tRNA standard (70-80
nucleotides) and two less prominent bands (most likely the 5S rRNA
and 5.8S rRNA) with mobilities slightly slower than the major band
of small RNA were detected on the 4% agarose gel. No bands of large
RNA or genomic DNA were detected.
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