QUANTITATION OF MESSENGER-RNA BY THE KINETIC POLYMERASE CHAIN-REACTION ASSAY - A TOOL FOR MONITORING P-GLYCOPROTEIN GENE-EXPRESSION

被引:94
作者
HOOF, T
RIORDAN, JR
TUMMLER, B
机构
[1] UNIV TORONTO,DEPT BIOCHEM & CLIN BIOCHEM,TORONTO M5G 1X8,ONTARIO,CANADA
[2] HOSP SICK CHILDREN,RES INST,TORONTO M5G 1X8,ONTARIO,CANADA
关键词
D O I
10.1016/0003-2697(91)90133-E
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The overexpression of P-glycoprotein (mdr1) induces the phenotype of multidrug resistance to many chemically unrelated drugs. Absolute mdr1 mRNA levels in tissues, neoplasms and cell lines from various rodents and man were estimated by primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labeled nucleotide. Aliquots taken during PCR were analyzed for product formation by two-dimensional densitometry of autoradiograms of separated cDNA on dried agarose gels. The kinetic RT/PCR assay was calibrated for the range of 10-3 to 103 amol of specific mRNA by utilizing mdr1 RNA transcribed from a full-length cDNA as the external standard and aldolase mRNA as the internal standard in order to assess RNA degradation of the specimen. For various cDNA fragments differing between 200 and 600 bp in size, the yield of cDNA during logarithmic growth increased by 1.78 ± 0.02 per cycle. Hence, the sensitive and reproducible quantitation of transcript is feasible by monitoring RT/PCR kinetics without the need for any competing cRNA or cDNA fragment provided that a high-performance thermal cycler is employed for PCR. © 1991.
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页码:161 / 169
页数:9
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