Journal
NATURE PROTOCOLS
Volume 4, Issue 3, Pages 356-362Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2009.8
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Funding
- DOE [DE-FG02-04ER15541]
- NSF [0445638, 0548569, 0321437]
- USDA [2007-01991]
- NIH [P20 RR16472-04]
- NATIONAL CENTER FOR RESEARCH RESOURCES [P20RR016472] Funding Source: NIH RePORTER
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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.
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