Schmittgen Thomas D, Jiang Jinmai, Liu Qian, Yang Liuqing
Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2004 Feb 25;32(4):e43. doi: 10.1093/nar/gnh040.
microRNAs (miRNAs) are small, functional, non-coding RNAs. miRNAs are transcribed as long primary transcripts (primary precursors) that are processed to the approximately 75 nt precursors (pre-miRNAs) by the nuclear enzyme Drosha. The approximately 22 nt mature miRNA is processed from the pre-miRNA by the RNase III Dicer. The vast majority of published studies to date have used northern blotting to detect the expression of miRNAs. We describe here a sensitive, high throughput, real-time PCR assay to monitor the expression of miRNA precursors. Gene-specific primers and reverse transcriptase were used to convert the primary precursors and pre-miRNAs to cDNA. The expression of 23 miRNA precursors in six human cancer cell lines was assayed using the PCR assay. The miRNA precursors accumulated to different levels when compared with each other or when a single precursor is compared in the various cell lines. The precursor expression profile of three miRNAs determined by the PCR assay was identical to the mature miRNA expression profile determined by northern blotting. We propose that the PCR assay may be scaled up to include all of the 150+ known human miRNA genes and can easily be adaptable to other organisms such as plants, Caenorhabditis elegans and Drosophila.
微小RNA（miRNA）是一类小的、具有功能的非编码RNA。miRNA最初转录为较长的初级转录本（初级前体），然后由核酶Drosha加工成约75个核苷酸的前体（前体miRNA）。约22个核苷酸的成熟miRNA由核糖核酸酶III Dicer从前体miRNA加工而来。迄今为止，绝大多数已发表的研究都使用Northern印迹法来检测miRNA的表达。我们在此描述一种灵敏、高通量的实时PCR检测方法，用于监测miRNA前体的表达。使用基因特异性引物和逆转录酶将初级前体和前体miRNA转化为cDNA。利用PCR检测法检测了六种人类癌细胞系中23种miRNA前体的表达。相互比较或在不同细胞系中比较单个前体时，miRNA前体积累到不同水平。通过PCR检测法确定的三种miRNA的前体表达谱与通过Northern印迹法确定的成熟miRNA表达谱相同。我们认为，PCR检测法可以扩大规模以涵盖所有150多种已知的人类miRNA基因，并且可以很容易地应用于其他生物体，如植物、秀丽隐杆线虫和果蝇。
