Cloning of Small RNAs for the Discovery of Novel MicroRNAs in Plants

Endogenous small RNAs can be grouped into several distinct classes of 21-nt-long microRNAs (miRNAs), short interfering RNAs (siRNAs), trans-acting siRNAs (tasiRNAs), and 24-nt long heterochromatic siRNAs. miRNAs are increasingly being recognized as significant effectors of gene regulation in a wide range of organisms. These molecules are typically ∼21-nt long and are amenable for cloning by streamlined protocols. Here we detail the methodology for cloning small RNAs in rice to identify novel miRNAs and other important small RNAs. Briefly, small RNA molecules are size fractionated, attached to adaptors, and subsequently converted into cDNA and PCR amplified. Current high-throughput sequencing technologies allow sequencing of the PCR products directly.