Gene dosage of different samples was performed with relative quantification real-time PCR method. Real-time PCR reaction was performed using Luna Universal qPCR Master Mix (NEB) and RPPH1 (a reference gene with a single copy) primers used for quantitative analysis were referred to Ahani's study [8 (link)]. Additionally, primers were designed for specific detection of CYP1B1 (exons 1 and 2), CYP2E1 (exons 8 and 9) and CYP2C9 (exons 4 and 7) (Table 1). The reaction was performed in 96 wells plate with 10 μL volume in total, which included 10 ng genomic DNA, 1X Luna Universal qPCR Mastermix (NEB), 0.25 μL for each primer (10 pmole/μL) and Ultrapure Distilled Water (ThermoFisher Scientific). Subsequently, the covered plates were run on LightCycler 96 Instrument (Roche) with the thermocycle with denaturation at 95°C for 10 min, following by 45 cycles (95°C for 15 s and 60°C for 60 s). The copy number of targeted exon in comparison to reference gene was determined according to the following equation: ΔΔCt = [CtRPPH1(Reference sample) − Ct targeted exon (reference sample)] − [CtRPPH1(Unknown sample) − Ct targeted exon (Unknown sample)]. The relative copy number of the genes was later calculated following the ratio equation (2−ΔΔCt).
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