Detection of multidrug-resistance mdr-1 gene expression using real-time RT-PCR

OBJECTIVE: To est-ablish a method for detection of the expression of mdr-1 gene in tumor cells via real-time RT-PCR.METHODS: The mdr-1 PCR product was subcloned into PGEM-T vector to contrust recombinant plasmid.After optimizing the thermcycling conditions,a quantitative RT-PCR assay to quantify mdr-1 gene expression was successfully developed on Line-gene real-time PCR detection system.RESULTS: The standard curve was successfully established and the linear relationship in statistics was good with the regression value 0.997.The detection limit of the real-time PCR assay was 102 copies of template in a 40 μL reaction volume.The intra-assay and inter-assay coefficients of variation were smaller than 1.0%.CONCLUSION: The method of detection of the expression of mdr-1 gene using standard curve constructed by TA cloning is easily performed and time-consuming,and has good reproducibility and reliability.