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.

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.