Schmittgen Thomas D, Lee Eun Joo, Jiang Jinmai, Sarkar Anasuya, Yang Liuqing, Elton Terry S, Chen Caifu
College of Pharmacy, Ohio State University, Columbus, OH 43210, USA.
Methods. 2008 Jan;44(1):31-8. doi: 10.1016/j.ymeth.2007.09.006.
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.
微小RNA（miRNA）通过聚合酶链反应（PCR）进行扩增具有挑战性，因为miRNA前体由稳定的发夹结构组成，而成熟的miRNA大小与标准PCR引物大致相同。尽管存在这些困难，但已经开发出成功的实时逆转录PCR技术来扩增和定量前体和成熟微小RNA。本文概述了我们开发的用于检测前体和成熟微小RNA的实时PCR技术。方案描述了使用相对（比较C(T)）和绝对（标准曲线）定量来呈现数据的方法。实时PCR测定法用于测量脂多糖刺激的单核细胞中前体和成熟miR-155表达的时间进程。提供了将测定配置为低密度PCR阵列以进行高通量基因表达谱分析的方案。通过对用12-O-十四烷酰佛波醇-13-乙酸酯诱导分化的HL60细胞中的200多种前体和成熟miRNA进行谱分析，有可能鉴定出在分化过程中其加工受到调控的miRNA。由于PCR的特异性和敏感性，实时PCR已成为核酸定量的金标准。技术进步使得对miRNA的定量成为可能，其质量可与更传统的RNA相媲美。
