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