Co-Authors:
German, M.A., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
Luo, S., Illumina Inc., 25861 Industrial Boulevard, Hayward, 94545, United States
Schroth, G., Illumina Inc., 25861 Industrial Boulevard, Hayward, 94545, United States
Meyers, B.C., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
Green, P.J., Delaware Biotechnology Institute, University of Delaware, Delaware, Newark, 19711, United States
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' doublestranded 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. © 2009 Macmillan Publishers Limited. All rights reserved.
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