NONRADIOACTIVE QUANTIFICATION OF MDR1 MESSENGER-RNA BY POLYMERASE CHAIN-REACTION AMPLIFICATION COUPLED WITH HPLC

We describe a new strategy for quantifying mRNA by using polymerase ch ain reaction (PCR) coupled with HPLC. PCR-amplified products are direc tly analyzed with a specific HPLC column and are quantified by standar dization against a housekeeping gene, beta-actin. To evaluate the expe rimental conditions, we examined the multidrug-resistance-associated m dr1 gene expression in two drug-sensitive cell lines (RPMI-8226 and KB -3-1) and in their drug-resistant sublines. This revealed a significan t overexpression of the mdr1 gene in chemoresistant sublines that para lleled the degree of in vitro drug resistance and the amounts of mRNA determined by other methods. Results were consistently reproducible, w ith imprecision (CV) <25%. The reverse transcription option PCR/HPLC p rotocol described here is a sensitive, nonradioactive method for quant ifying mdr1 mRNA in cell lines or clinical tumor samples, and can be a pplied to the simultaneous quantification of several mRNA species.