[Construction of the internal standard RNA and DNA templates for MRP gene quantitative RT-PCR analysis]

To construct the internal standard RNA and DNA templates for quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitatively measure the mRNA expression of MRP gene.

A 292 bp fragment was amplified by PCR from plasmid pRC/RSV-MRP containing full-length MRP cDNA and inserted into the Sma I site of PUC19 vector, constructing a recombinant plasmid-pUMRP292. A 238 bp HGV fragment was amplified by PCR from plasmid pUHGV and inserted into the Cla I site of pUMRP292, constructing a new recombinant plasmid-pUMRP 292/HGV as the internal DNA competitive template for quantitative PCR of MRP. A 530 fragment containing the 292 bp of MRP and the 238 bp of HGV was cut down by EcoR I and Xba I pUMRP292/HGV and cloned into the transcriptional vector pSP72 by the same enzyme sites, constructing a recombinant plasmid-pSMRP292/HGV, and then pSMRP292/HGV was cleaved with EcoR V and transcripted in vitro by SP6 RNA polymerase, obtaining a 530 bp mutant MRP-RNA positive strand as the internal RNA competitive template for quantitative RT-PCR.

The established quantitative RT-PCR assay is simple, rapid and sensitive, and the DNA competitive template is more simple, economic and reliable than the RNA one.