German Marcelo A, Luo Shujun, Schroth Gary, Meyers Blake C, Green Pamela J
Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.
Nat Protoc. 2009;4(3):356-62. 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 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.
我们开发了一种名为RNA末端平行分析（PARE）的新方法，用于高通量鉴定微小RNA（miRNA）靶标以及在RNA降解组研究中的多种应用。这里描述的方法包括改良的5'-cDNA末端快速扩增、mRNA 3'切割产物的深度测序和生物信息学分析。在RNA提取和聚腺苷酸化RNA分离后，将包含MmeI识别位点的5'-RNA接头连接到5'-单磷酸化产物上，这些产物包含通过miRNA诱导切割产生的mRNA片段。连接产物进行逆转录、轻微扩增并用MmeI切割。5'等长片段通过凝胶选择，连接到3'双链DNA接头上并进行PCR扩增。经过凝胶纯化后，产物进行深度测序。然后将数据与cDNA匹配并通过生物信息学筛选进行分析。我们详细描述了高通量方案，并指出了PARE的其他用途。这里介绍的程序可以在6-7天内完成。
