2010高级分子生物学专题-第五讲-小RNA定量.pdfVIP

Available online at Methods 44 (2008) 31–38 /locate/ymeth Real-time PCR quantification of precursor and mature microRNA Thomas D. Schmittgen a,*, Eun Joo Lee a, Jinmai Jiang a, Anasuya Sarkar b, Liuqing Yang a, Terry S. Elton a,b, Caifu Chen c a College of Pharmacy, Ohio State University, Columbus, OH 43210, USA b Heart and Lung Research Institute, Ohio State University, Columbus, OH, USA c Applied Biosystems, Foster City, CA, USA Accepted 21 September 2007 Abstract microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative CT) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who’s processing is regulated duri