Abstract

We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5|[prime]|-rapid amplification of cDNA ends, deep sequencing of 3|[prime]| cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5|[prime]|-RNA adapter that includes an MmeI recognition site is ligated to 5|[prime]|-monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5|[prime]| equally-sized fragments are gel-selected, ligated to a 3|[prime]| double-stranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high-throughput protocol in detail and indicate alternative uses for PARE. The procedure presented here can be accomplished in 6–7 d.