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.
Yang
Z
Z
University of Washington, Seattle 98195-7610, USA.
Woodahl
E L
EL
Wang
X Y
XY
Bui
T
T
Shen
D D
DD
Ho
R J Y
RJ
D002460:Cell Line; D005786:Gene Expression Regulation; D019450:Genes, MDR / Q000235:genetics*; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D008214:Lymphocytes; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger