改进茎环引物RT-PCR法实时定量检测microRNA

Real-time Quantification of MicroRNA by Improved Stem-loop Primer RT-PCR

目的:为检测不同组织中microRNA(miRNA)的表达量,本研究建立了一种改进的qRT-PCR(Quantitative reverse transcription PCR)技术。方法:采用具有高特异性的茎环引物逆转录,结合SYBR Green实时定量PCR技术,建立了以延伸茎环引物RT-PCR为基础的实时定量检测microRNA的方法。结果:本研究建立的microRNA检测方法,特异性好,灵敏度高,线性范围宽。熔解曲线的单一峰型和电泳检测到的单一条带都证明了扩增的特异性。对标准品的检测跨越了7个数量级的浓度范围,最低拷贝数为103,效率高达98%。结论:利用该技术检测了120个组织样本,说明该方法可快速、准确、高效的获取不同组织中miRNA的表达量,为进一步探讨miRNA对性发育的影响提供了实验依据。

Objective： To establish an improved qRT-PCR （Quantitative reverse transcription PCR） technology to detect the expression levels of microRNA （miRNA） in different tissues. Methods： Reverse transcription using high specific stem-loop primer and amplified by SYBR Green real-time PCR, established real-time quantitative method of miRNA based on the stem-loop primer extension RT-PCR. Results This method was specificity, sensitivity and had a high dynamic range. A single dissociation peak on the thermal melting curve and a single DNA band on agarose gel signified the amplification reliability and specificity for miRNA. It exhibited a dynamic range of seven orders of magnitude from 103 to 109, efficiency was up to 98 %. Conclusion： This assay is a fast, accurate and efficient method to quantification of miRNA indicated by detection of 120 tissue samples, provides an experimental basis for further investigate the effects of miRNA on sexual development.