Semi-quantitative RT-PCR method to estimate full-length mRNA levels of the multidrug resistance gene

Expression levels of P-glycoprotein (P-gp), the transporter encoded by the human multidrug resistance gene (MDR1), may play an important role in drug disposition. The ability to quantitate full-length MDR1 mRNA levels may be predictive of P-gp expression and function. Therefore, a semi-quantitative RT-PCR assay was developed to assess full-length MDR1 mRNA levels. Levels offull-length 3.8-kb MDR1 mRNA were estimated by comparing PCR amplification of the RNA extract with that of an internal standard, deltaMDR1. The 2.9-kb deltaMDR1 competitor RNA standard was constructed by deleting 965 bpfrom the interior of MDR1 mRNA. The full-length MDR1 and deltaMDR1 share identical 5' and 3'primer binding sequences, allowing for their simultaneous amplification in the same RT-PCR. With this approach, MDR1 mRNA levels can be sensitively and reliably estimated with a detection limit of 2000 copies. Full-length MDR1 mRNA levels in various human cell lines and lymphocytes from leukemia patients varied over 100-fold, ranging from 0.3 to 36.5 x 10(5) copies/microg total RNA. The semi-quantitative full-length RT-PCR assay may be useful in estimating MDR1 mRNA levels to assess P-gp expression, which may be important in studying the role of P-gp in drug disposition and cancer chemotherapy efficacy.