title: Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome.
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.
authors.affiliation: Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA. mgerman@dbi.udel.edu
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: German
authors.forename: Shujun
authors.initials: S
authors.lastname: Luo
authors.forename: Gary
authors.initials: G
authors.lastname: Schroth
authors.forename: Blake C
authors.initials: BC
authors.lastname: Meyers
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Green
mesh_terms: D000818:Animals; D019295:Computational Biology / Q000379:methods*; D035683:MicroRNAs / Q000235:genetics*; D020871:RNA Stability; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D035683:MicroRNAs