Construction of Parallel Analysis of RNA Ends (PARE) Libraries for the Study of Cleaved MiRNA Targets and the RNA Degradome

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2009 Feb 28

PMID 19247285

Citations 170

Authors

Marcelo A German

Shujun Luo

Gary Schroth

Blake C Meyers

Pamela J Green

Affiliations

Soon will be listed here.

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'-rapid amplification of cDNA ends, deep sequencing of 3' cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5'-RNA adapter that includes an MmeI recognition site is ligated to 5'-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' equally-sized fragments are gel-selected, ligated to a 3' 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.

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