[Development, optimization and application of the expression analysis platform based on multiplex quantitative RT-PCR using fluorescent universal primers].
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.
Wang
Qin-Xi
QX
Institute of Biological Science & Biotechnology, Donghua University, Shanghai 201620, China. wqx457@163.com
Li
Kai
K
Zhou
Yu-Xun
YX
Xiao
Jun-Hua
JH
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D005456:Fluorescent Dyes; D015870:Gene Expression; D020869:Gene Expression Profiling; D008297:Male; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012333:RNA, Messenger