Detection Of Multidrug-Resistance Mdr-1 Gene Expression Using Fluorescent Quantitative RT-PCR

[Objective] To establish a rapid and sensitive method for detecting the expression of multidrug resistance mdr-1 gene in tumor cells by fluorescent quantitative RT-PCR(FQ-RT-PCR). It will have great help in clinical therapy and judgement of the tumor prognosis.[ Materials and Methods ]1. Cell cultivation: The breast cancer cell lines MCF-7/ADR were routinely grown in RPMI1640 cultivation medium containing 10% fetal calf serum, penicllin and streptomycin in a humidified incubator at 37℃ and 5% CO₂.2. Extraction of total RNA: The total RNA of breast cancer cell lines MCF-7/ADR and 58 primary breast tumor samples were extracted with Biozol respectively according to the manufacturer's instructions.3. Reverse Transcription: Reverse transcription was performed using total RNA with Oligo dT as a template and M-MLV Reverse Transcriptase in a sterile RNase-free microcentrifuge tube.4. Polymerase Chain Reaction and Consruction of recombined plasmid of mdr-1: PCR amplification used primers specific for mdr-1 was performed . The PCR product was gel-purified and subcloned into the PGEM-T Easy vector according to the protocol. The recombinant plasmid was transformed into E.coli.JM109 competent cells and sequence-verified clones were selected through blue-white screening .