Protocol abstract
Nature Protocols 4, - 356 - 362 (2009)
Published online: 26 February 2009 | doi:10.1038/nprot.2009.8
Subject Categories: Computational and theoretical biology | Genetic analysis | Genomics and proteomics | Nucleic acid based molecular biology
Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome
Marcelo A German1, Shujun Luo2, Gary Schroth2, Blake C Meyers1 & Pamela J Green1
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.
- Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, USA.
- Illumina Inc., 25861 Industrial Boulevard, Hayward, California 94545 USA.
Correspondence to: Marcelo A German1 e-mail: mgerman@dbi.udel.edu
Correspondence to: Pamela J Green1 e-mail: green@dbi.udel.edu
nature-products
A-Z product listing
- MmeI(New England Biolabs)
- 0.1 M DTT(Invitrogen)
- 10 bp DNA ladder(Invitrogen)
- 10
SAM(New England Biolabs) - 10 T4 RNA ligase buffer(Ambion)
- 3 M NaOAc(Ambion)
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