METHODS FOR CLONING SMALL RNA SPECIES

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.