Development, Optimization And Application Of The Expression Analysis Platform Based On Multiplex Quantitative RT-PCR Using Fluorescent Universal Primers

Multiple gene expression analysis is an an effective way to screen candidate genes and identify gene function on transcriptional level. Currently, there is still a niche for qutitative moderate-throughput （10～30 genes） expression analysis methods in all commonly used methods, including cDNA array which is high-throughput but lacks in quatitave capability, and real-time qPCR on the contrary. A multiplex quantitative RT-PCR technology with a universal fluorescent primer was established. It employs a chimeric-primer-induced-universal-primer amplification method that ensures target genes amplified in a constant ratio during the reaction. In the first few cycles, two gene-specific primers, which carry on the 5’ ends a universal sequence, are used to amplify the specific gene targets. The resulting PCR templates are tailed with the universal sequences. As the reaction proceeds, universal primers, present at high concentrations in the reaction mixture, take over the amplification process. The transition from using many different primers to just two effectively collapses the reaction complexity, as all the products are treated as the same chemical species and not differentially amplified. 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 qPCR with low throughput, through improving the entire process of expression profiling analysis.Previous study has found a QTL segment regulating mouse puberty onset on Chromosome X by whole genome scanning and modified interval-specific congenic strain analysis. In this study, eleven genes within this QTL segment were investigated to construct and optimize the method, which includs the following thress aspects. Unviversal primers and 14 pairs of chimeric primers （including three housekeeping genes’） were designed; the concentration of reverse chimeric primers and reaction time was investigated during the reverse transcription reaction; the concentration ratios of universal primers and chimeric forward primers, reaction conditions (such as Mg²⁺ concentration, annealing and extension time), the method of forward chimeric primers touchdown PCR and universal primers addition , and test sensitivity were determined, in the PCR reaction course.As the results showed, the sensitivity of detection （10² copies） was determined, the ratio of universal primers’ concentration and chimeric forward primers’ （1:1） was optimized, and the accuracy and repeatability were validated. Amplification of gene expression in low abundance was enhanced with forward chimeric primers touchdown PCR and universal primers addition. Finally, by 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 days, one differentially expressed gene PHF6 was found for further function determination.