Cloning of small RNA molecules.

Small RNAs that are derived from dsRNA precursors act as guide RNAs during sequence-specific epigenetic regulation of eukaryotic gene expression. These small regulatory RNAs are between 20 and 30 nucleotides in length, and fall into one or more of the following categories: small interfering RNAs (siRNAs), microRNAs (miRNAs), and heterochromatic siRNAs (hsiRNAs). Procedures to record the profile of small RNAs expressed in cultured cells or tissues are described. The small RNAs are directionally cloned after isolation from total RNA. The methods rely on T4 RNA ligase-based joining of adapter oligonucleotides to the 3' and 5' termini of the pool of small RNAs. The ligation products are reverse transcribed and PCR-amplified. It is recommended to directionally concatamerize the relatively short PCR products before cloning in order to increase the number of RNA sequences obtained per clone.