Methods and compositions for the rapid isolation of small RNA molecules
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.
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.