MicroRNAs (miRNAs) are an important class of small RNAs that regulate protein translation (see Getting Started with MicroRNA Investigation ). To date, 230 mammalian miRNAs have been identified and cataloged in the Sanger miRNA Registry. Some scientists believe that the number of miRNAs could exceed 1000 [1]. While cloning and sequencing were used to identify most of the known mammalian miRNAs, increasing effort is being put into bioinformatic prediction of new miRNA sequences. A combination of computational biology tools and molecular cloning methods will likely be used for characterizing additional miRNAs.

Isolating the mature miRNA fraction is the first critical step in cloning these small regulatory molecules. Ambion scientists have developed two sequential systems that are optimized for isolating and enriching RNA preparations that contain 17�24 nucleotide, mature miRNAs. Here we describe the process by which miRNAs can be cloned in an effort to identify new members of this small RNA family.

1. Isolate total RNA using a method that will capture small RNAs

•mir Vana miRNA Isolation Kit includes small RNAs in the total RNA preparation and can subsequently be used to enrich for RNA <200 nt

2. Separate mature miRNAs from precursor molecules

•flashPAGE Fractionator System will quickly and easily purify mature miRNA

3. Ligate 3'' donor oligonucleotide to RNA with T4 RNA ligase

•The donor oligonucleotide may contain deoxynucleotides or both ribonucleotides and deoxynucleotides

•This donor oligonucleotide should contain a 3'' end blocking group (e.g., amino group, adenylation) to prevent self ligation and ligation to the RNA 5'' ends

•The donor oligonucleotide should be 5'' phosphorylated

•The donor oligonucleotide should contain a restriction enzyme site that is compatible with your cloning vector

•Example: 5''-pCTGTAGGCACC ATCAAx-3'' [2] (p=phosphorylation, bold type=restriction site, x=blocking group)

4. Gel purify RNA

•Remove excess 3'' oligonucleotides, enzymes, and salts

5. Ligate 5'' acceptor oligonucleotide to RNA with T4 RNA ligase

•This acceptor oligonucleotide will contain both ribonucleotides and deoxynucleotides

•The acceptor oligonucleotide should contain the same restriction enzyme site that is in the 3'' donor oligonucleotide

•Example: 5''-ATCGTaggcacc tgaaa-3'' [2] (upper case=DNA, lower case= RNA, bold type=restriction site)

6. Gel purify RNA

•Remove excess 5'' oligonucleotides, enzymes, and salts

7. Use RT-PCR to amplify RNA

• RT and PCR primers are based on acceptor and donor oligonucleotide sequences used above

8. Digest with restriction enzyme whose target site was incorpo-
  rated into donor and acceptor oligonucleotides

9. Concatamerize PCR products with T4 DNA ligase and fill in the
  ends of the PCR products

10. Isolate ligation products (400�600 nt range) by gel purification

•Resulting clones contain 4�6 inserts and provide longer templates for efficient sequencing analysis

11. Clone into your cloning vector of choice

12. Screen for vectors containing inserts by PCR

13. Purify vector/insert and sequence inserts

Additional details have been described by leading miRNA researchers [2�5].

Summary

This is one method that has been used to uncover new miRNAs. When moving into validation and functional studies, we recommend using Pre-miR miRNA Precursor Molecules and Anti-miR miRNA Inhibitors to alter miRNA activity in a cell system. See www.ambion.com/miRNA for more information.