遗传 ›› 2009, Vol. 31 ›› Issue (5): 552-552―560.doi: 10.3724/SP.J.1005.2009.00552

• 技术与方法 • 上一篇    

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

王勤熙;李凯;宇荀;肖君华   

  1. 东华大学生物科学与技术研究所, 上海201620
  • 收稿日期:2008-11-04 修回日期:2009-02-10 出版日期:2009-05-10 发布日期:2009-05-10
  • 通讯作者: 李凯

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

WANG Qin-Xi;LI Kai;ZHOU Yu-Xun;XIAO Jun-Hua   

  1. Institute of Biological Science & Biotechnology, Donghua University, Shanghai 201620, China
  • Received:2008-11-04 Revised:2009-02-10 Online:2009-05-10 Published:2009-05-10
  • Contact: LI Kai

摘要: 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。

关键词: 基因表达谱分析, 性发育启动, 多重定量RT-PCR, 通用引物

Abstract: A multiplex quantitative RT-PCR technology with a universal fluorescent primer was established. This tech-nology 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 through-put, 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 sig-nificantly 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.