Stem-loop qRT-PCR for the detection of plant microRNAs

Abstract

Plant microRNAs (miRNAs) play important roles in the posttranscriptional regulation of protein-coding genes, and they are essential for a normal development and survival. Mature miRNAs are cleaved from larger precursor RNAs and are typically 21-22 nt long. The small size, the lack of a common feature like a poly(A) tail, 3′ end-modifications, and presence of a precursor—all these factors affect the detection and hinder the quantification of miRNAs. The stem-loop qRT-PCR method described here is designed to detect and quantify mature miRNAs in a fast, specific, accurate, and reliable manner. Firstly, a miRNA-specific stem-loop RT primer is hybridized to miRNA and then reverse transcribed. Next, the RT product is amplified and monitored in real time using a miRNAspecific forward primer and a universal reverse primer. This method enables miRNA expression profiling from as little as 10 pg of total RNA, and it is suitable for a relatively high-throughput analysis of miRNA expression.