两种实时定量RT-PCR方法检测miRNAs表达的技术分析

Technical analysis for detection and quantification of microRNAs by two real-time quantitative reverse transcription methods

目的比较两种实时定量反转录聚合酶链反应(RT-qPCR)方法检测microRNAs(miRNAs)表达含量的技术差异,优化不同实验目的和条件下研究miRNAs表达的技术方法。方法取21d和42d两个时间点的SD大鼠关节软骨组织,采用Trizol法提取总RNA备用。选取rno-miR-15b、rno-miR-16、rno-miR-195、rno-miR-497作为研究对象,分别用茎环引物和试剂公司提供的试剂盒方法反转录总RNA,并应用实时定量PCR方法检测这些miRNAs的表达量。提取人血浆中总RNA,用上述两种RT-qPCR方法实时定量检测has-miR-16的表达量。结果两种方法检测这些miRNAs表达量,在大鼠21d和42d这两个时间点其表达量变化趋势相同,都呈现增高的趋势,这与我们前期Solexa测序结果相同。在血浆中的结果显示,其中茎环引物反转录方法灵敏度相对较高。结论茎环引物法在少量几个重要的miRNAs检测中具有优势,而试剂盒方法适用于大量miRNAs的筛查。

Objective To optimize the method for quantifying microRNAs in different experimental purposes and conditions by comparing two real-time quantitative reverse transcription-polymerase chain reaction （RT-qPCR） methods. Methods We isolated the total RNA from the SD rat articular cartilage at postnatal day 21 and day 42 with TRIzol reagent. The RT reactions were performed by stem-loop primers and the universal primer of miRNA detection kit respectively; then real time PCR was performed to test the expressions of rno-miR-15b, rno-miR-16, rno-miR-195 and rno-miR-497. In addition, the total RNAs in human plasma were isolated by using TR1 Reagent BD （MRC, TR126） according to the instructions by the manufacturer with two different RT-qPCR methods to quantify the expression of has-miR-16. Results The expression change of these miRNAs was of the same increase trend by the two different RT-qPCR methods, which accorded with the results of our Solexa sequencing. The results of plasma demonstrated that stem-loop RT-qPCR method was relatively sensitive. Conclusion Stem-loop RT- qPCR method shows the advantages of testing a few important miRNAs while the detection kit method applies to screening a large number of miRNAs.