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


RESULTS
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.


CONCLUSIONS
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.
Department of Hematology, Liuhua Bridge Hospital, Guangzhou 510010.
L
L
Cao
B
B
Wang
Z
Z
Yu
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D004247:DNA / Q000235:genetics; D019450:Genes, MDR / Q000235:genetics*; D006801:Humans; D007938:Leukemia / Q000235:genetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013698:Templates, Genetic
D018528:ATP-Binding Cassette Transporters; D004247:DNA; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins