茎-环RT-PCR法定量miRNA-421的引物设计

Stem-Loop Real-Time Quantitative PCR for Quantification of miRNA-421 by Specific Primers

本研究旨在通过茎-环RT-PCR法,建立有效定量miRNA-421的PCR方法,探讨该方法在定量检测miRNA方面的应用价值.利用miRBase数据库获得miRNA-421的成熟序列.设计3条miRNA-421特异性反转录引物,Q-PCR上游引物及通用下游引物.通过10倍梯度稀释RNA后特异性反转录,RT-PCR分析不同引物互相配对PCR的扩增曲线及熔融曲线,鉴定出特异性好、扩增效率高的引物.为进一步鉴定引物的有效性,过表达miRNA-421,用鉴定的成熟引物定量扩增.利用茎-环RT-PCR法设计、鉴定出miRNA-421引物:421RT7、F19,且这对引物特异性好、扩增效率高.通过设计引物组合,茎-环RT-PCR法能够很好地用于miRNA定量分析,并具有准确、快速、节约成本的优点.

This study was to establish a method of quantitative detecting miRNA-421 by stern-loop RT-PCR, and to eval- uate the feasibility of this method in quantitative assay miRNA, miRBase database was used to get the mature sequence of miRNA-421. 6 primers were designed, including 3 primers for miRNA-421 specific reverse transcription, 2 forward and 1 general reverse primers for quantitative PCR, respectively. Different RT primers and forward primers were paired in Q-PCR. RNA after 10 times dilution was reverse transcribed and then detected by Q-PCR. Amplification curves and melting curves were analyzed to identify the primers with high specificity and efficiency of amplification. In order to fur- ther validate this primer, miRNA-421 was over-expressed in HepG2 and detected by Q-PCR. 2 primers, 421RT7 and 421F19 with high specificity and efficiency of amplification were obtained using stem-loop RT-PCR. Stem-loop RT-PCR provided a method that enabled fast, accurate and sensitive detection of miRNA.