基于荧光通用引物的多重定量RT-PCR基因表达谱分析平台的构建、优化及其应用

Development, optimization and application of the expression analysis platform based on multiplex quantitative RT-PCR using fluorescent universal primers

文章建立并优化了一种基于荧光通用引物的多重定量RT-PCR技术,该技术采用了嵌合特异引物引导荧光通用引物的扩增方案,多个目的基因被一对通用引物等比例扩增,从而实现多重定量检测。该技术实现了经济可靠的中通量基因表达定量研究,弥补了基因表达分析平台中cDNA芯片定量准确性低和Real-time quanti-tative PCR通量小的缺点,完善了整个基因表达的分析过程。文章以小鼠X染色体上影响性发育启动的QTL区段为例,选择11个目的基因进行了技术构建及优化,确定了该技术的检测灵敏度为102拷贝,通用引物与上游嵌合特异引物的比例以1:1为佳,并且验证了该技术的重复性和准确性。降落式(Touchdown)PCR结合通用引物补加实验表明,该优化步骤可大大改善低丰度表达基因的扩增。通过对2个品系(C3H/HeJ和C57BL/6J)15日龄小鼠的下丘脑和睾丸组织中的11个基因的表达分析,在下丘脑中找到了一个差异表达的基因PHF6可用于进一步的基因功能研究。

A multiplex quantitative RT-PCR technology with a universal fluorescent primer was established. This technology employs a chimeric-primer-induced-universal-primer amplification method that ensures target genes amplified in a constant ratio. This technique was cost-effective, moderate-throughput, and reliable in quantification of gene expression. It is complementary to cDNA chip, which has low quantitative accuracy, and Real-time quantitative PCR with low throughput, through improving the entire process of expression profiling analysis. Eleven genes within a QTL segment regulating mouse puberty onset on chromosome X were investigated to construct and optimize the method. The sensitivity of detection （102 copies） was determined, the concentration ratio of universal primer and chimeric forward primers （1：1） was optimized, and the accuracy and repeatability were validated. The method of Touchdown PCR with addition of universal primers significantly improved amplification of genes expressed in low abundance. After testing the expression profile of 11 genes in hypothalamus and testis in two mouse strains C3H/HeJ and C57BL/6J at the age of 15 d, one gene named PHF6 was found differentially expressed for further function analysis.