2种微小RNA实时定量PCR检测方法的比较

目的:对目前最常用的检测微小RNA(miRNA)的茎环实时定量PCR法和PAP实时定量PCR法进行比较。方法:分别用茎环实时定量PCR法和PAP实时定量PCR法检测人乳腺癌细胞MCF-7中U6和23种miRNA的表达,利用定量PCR分析软件和琼脂糖凝胶电泳方法,将2种方法在引物设计难度、特异性与灵敏度,以及检测通量方面进行比较。结果:茎环法的特异性和灵敏度比PAP法高,但引物设计难度大,检测通量低;PAP法引物设计难度较低,检测通量较高,但特异性和灵敏度较差。结论:茎环法实时定量PCR适于有针对性地检测小规模miRNA,而PAP法则适于大规模miRNA筛选实验。

Comparison of Two Methods for Detection of microRNA by Real-Time Quantitative PCR

Objective：We aim to compare two method＇s performance on detecting microRNA（miRNA） expression level.The two methods are stem-loop real-time quantitative PCR（stem-loop qPCR） and PAP real-time quantitative PCR（PAP qPCR）.Methods： We invastigated the exprssion of U6 and twenty-three miRNAs in MCF-7 cells by stem-loop qPCR and PAP qPCR.Four characters of detection performance,which includes primer designing,speci-ficity,sensitivity and throughput,have been assessed by analysis software of real-time quantitative PCR and agarose gel electrophoresis.Results： Compared with the PAP qPCR,the stem-loop qPCR is a more specific and sensetive detective method,although its primer design is more sophisticated and throughput is lower.Conclusion：The stem-loop qPCR is suitable for small-scale detection of miRNAs,and the PAP qPCR is suitable for large scale screening of miRNAs experiment.