两种实时定量RT—PCR方法检测miRNAs表达的技术分析

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rnomiR-15b、rno—miR16、rrlo—miR195、rnomiR-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—miR16的表达量。结果两种方法检测这些miRNAs...

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Published in西安交通大学学报（医学版） Vol. 34; no. 2; pp. 258 - 262
Main Author 闵自信 杜小云 宁启兰 钟楠楠 郑悦雯 韩燕 吕社民 张蕊
Format Journal Article
LanguageChinese
Published 西安交通大学医学院遗传学与分子生物学系,陕西西安,710061 2013
Subjects
miRNAs
实时定量反转录聚合酶链反应
茎环引物
血浆
软骨
软骨
miRNAs
茎环引物
实时定量反转录聚合酶链反应
血浆
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Summary: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rnomiR-15b、rno—miR16、rrlo—miR195、rnomiR-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—miR16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。
Bibliography: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 meth
ISSN:1671-8259