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Nat Protoc. 2009;4(3):356-62. doi: 10.1038/nprot.2009.8.

Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome.

Author information

1
Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA. mgerman@dbi.udel.edu

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.

PMID:
19247285
DOI:
10.1038/nprot.2009.8
[Indexed for MEDLINE]

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