Methods for cloning small rna species

Abstract

This invention pertains to methods for cloning microRNA (miRNA) and other small ribonucleic acid (RNA) species from relevant cell sources.

1 . A method for identifying a desired fragment size during RNA size purification, said method comprising:
 providing a control RNA, wherein the control RNA is of a size that corresponds to the desired fragment size, wherein the control RNA contains a 3′-OH and does not contain a 5′ phosphate group, mixing said control RNA with a natural RNA sample, performing size separation on said mixture, and utilizing the control RNA to identify the location of a desired species within the unknown sample.   
     
     
         2 . The method of  claim 1  wherein the control RNA is distinct from known RNA species. 
     
     
         3 . A method according to  claim 1  wherein the desired fragment size is 19-25 bases in length. 
     
     
         4 . A method according to  claim 2 , wherein the control RNA is SEQ ID NO: 1. 
     
     
         5 . A method according to  claim 1  wherein the desired fragment size corresponds is 26-32 bases in length. 
     
     
         6 . A method according to  claim 2  wherein the control RNA is SEQ ID NO:2. 
     
     
         7 . A method according to  claim 1  wherein the control RNA has a length that is within four nucleotides of the desired fragment size. 
     
     
         8 . A method for recovery of a desired RNA fragment from a denaturing PAGE using a spin column dye terminator removal (DTR) column, said method comprising:
 a) separating a total RNA sample on a denaturing PAGE;   b) staining the PAGE gel with a nucleic acid stain and placing the gel on a long wavelength UV light box;   c) selecting a gel fragment containing the desired RNA fragment to be purified and excise from the gel;   d) crushing the gel fragment and adding said gel fragment to a spin column or DTR column;   e) discarding the spin column or DTR column; and   f) spinning the solution to separate a supernatant from the desired RNA fragment.   
     
     
         9 . A method for performing chemical adenylation on an oligonucleotide on a support, said method comprising:
 a) providing a support-bound oligonucleotide having a free 5′ hydroxyl group;   b) phosphitlyating the oligonucleotide with a diphenyl phosphate in an acetonitrile/pyridine/N-methylimidazole solution;   c) rinsing with a pyridine/acetonitrile solution;   d) converting the phosphitylated oligonucleotide to a phosphite triester with chlorotrimethylsilane; and   e) treating the phosphate triester with between a 30 and 50 molar excess of adenosine monophosphate in a pyridine/N-methylimidazole solution.   
     
     
         10 . A composition for ensuring the selection of a desired fraction of RNA corresponding to microRNA during RNA purification, said composition comprising SEQ ID NO:1. 
     
     
         11 . A composition for ensuring the selection of a desired fraction of RNA corresponding to PIWI-interacting RNA during RNA purification, said composition comprising SEQ ID NO:2. 
     
     
         12 . A kit for cloning small RNA species, said kit comprising:
 a) a 3′ cloning linker oligonucleotide;   b) a 5′ cloning linker oligonucleotide;   c) an internal control RNA;   d) a ligation enhancer;   e) a T4 RNA ligase; and   f) a T4 DNA ligase.   
     
     
         13 . A kit according to  claim 12  wherein the kit also comprises:
 a) a forward PCR primer;   b) a reverse transcription/PCR primer;   c) water that is substantially free of RNase, DNase and pyrogen;   d) Tris/EDTA buffer;   e) a ligation buffer;   f) ATP;   g) Glycogen; and   h) Sodium acetate.