Real-time PCR quantification of precursor and mature microRNA
Real-time PCR quantification of precursor and mature microRNA
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DOI：
10.1016/j.ymeth.2007.09.006
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发表时间：
2008-01-01
期刊：
影响因子：
4.8
通讯作者：
Chen, Caifu
中科院分区：
文献类型：
--
作者：
Schmittgen, Thomas D.;Lee, Eun Joo;Chen, Caifu
MicroRNAs(MiRNAs)是一种具有挑战性的分子，因为miRNA的前体由稳定的发夹组成，而成熟的miRNA的大小大致相当于标准的PCR引物。尽管有这些困难，但成功的实时RT-PCR技术已经被开发出来，以扩增和量化前体和成熟的microRNA。本文对我们开发的检测前体和成熟microRNAs的实时荧光聚合酶链式反应技术进行了综述。协议使用相对(比较C-T)和绝对(标准曲线)量化来描述数据的表示。实时荧光定量聚合酶链式反应检测内毒素刺激单核细胞中miR-155前体和成熟蛋白表达的时程。提供了将分析配置为用于高通量基因表达谱的低密度PCR阵列的协议。通过对12-O-十四烷基佛波醇-13-乙酸酯诱导分化的HL60细胞中200多个前体和成熟miRNAs的分析，有可能确定哪些miRNAs的加工在分化过程中受到调控。由于实时荧光定量聚合酶链式反应的特异性和敏感性，它已成为核酸定量的金标准。技术进步使miRNA的量化成为可能，其质量可与更传统的RNA相媲美。(C)2007 Elsevier Inc.保留所有权利。
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 C-T) 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 during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs. (c) 2007 Elsevier Inc. All rights reserved.