Identification of plant microRNAs using expressed sequence tag analysis.

microRNAs (miRNAs) are a new class of small endogenous noncoding regulatory RNAs, which play an important function in plant growth, development, phase change, and response to environmental stress. Identifying miRNAs is the first step for investigating miRNA-mediated gene regulation and miRNA function. In this chapter, we describe a comprehensive comparative genomics-based expressed sequence tag (EST) analysis for identifying miRNAs from a wide range of plant species. EST analysis is based on the conservation of miRNA sequences and the stem-loop hairpin secondary structures of miRNAs. In this method, potential miRNAs will first be identified by EST analysis followed by confirmation using TaqMan(®) MicroRNA qRT-PCR. This method is simple and reliable with high efficiency. This method has also been widely adopted by many scientists around the world and several hundreds of miRNAs have been identified in many plant species using this method.