西安交通大学学报（医学版）2013，Vol.34Issue(2)：258-262,5.
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
摘要
Abstract
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(R) 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 TRI 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.关键词
miRNAs/茎环引物/实时定量反转录聚合酶链反应/软骨/血浆Key words
miRNAs/ stem-loop primer/ real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR)/ cartilage/ plasma分类
医药卫生引用本文复制引用
闵自信,杜小云,宁启兰,钟楠楠,郑悦雯,韩燕,吕社民,张蕊..两种实时定量RT-PCR方法检测miRNAs表达的技术分析[J].西安交通大学学报（医学版）,2013,34(2):258-262,5.基金项目
国家自然科学基金资助项目(No.81170017) （No.81170017）
