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

• Published in: Nature protocols Vol. 4; no. 3; pp. 356 - 362
• Main Authors: German, Marcelo A, Green, Pamela J, Luo, Shujun, Schroth, Gary, Meyers, Blake C
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.02.2009; Nature Publishing Group
• Subjects: Analytical Chemistry; Animals; BASIC BIOLOGICAL SCIENCES; Bioinformatics; Biological Techniques; Biomedical and Life Sciences; Computational Biology - methods; Computational Biology/Bioinformatics; Deoxyribonucleic acid; DNA; Filters; Genetic research; Life Sciences; Methods; Microarrays; MicroRNAs; MicroRNAs - genetics; Organic Chemistry; Physiological aspects; Polymerase chain reaction; protocol; RNA; RNA Stability; Sequence Analysis, RNA - methods; United States; United States > US
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2009.8

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 Mme I 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 Mme I. 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.