实时荧光定量PCR技术的操作实践

摘要

This paper takes the relative quantitative method of SYBR? Green I as an example,designing an experimet whose aim is to detect the gene silencing effect of HIV-1 vpr specific siRNAs with model Mx3000P instrument. How to prepare the samples and how to set up the detection program and parameters were presented. The analysis method was introduced with the use of dissociation cure and amplification plots. The importance of overall experiment design, primer design,the optimization of PCR reaction system and appropriated internal control was emphasized.%采用Mx3000P型实时荧光定量PCR仪,以SYBR(R) Green I法相对定量技术为例,设计1例检测针对HIV-1 vpr基因的特异性siRNA基因沉默效果的实验.介绍了实时荧光定量PCR技术上机前样本的制备过程、检测程序及相关参数的设置方法等操作流程；结合熔解曲线、扩增曲线进行结果分析；强调了实验整体设计、引物设计与PCR反应体系的优化和内参的恰当选择在实时荧光定量PCR技术中的重要性.