Taqman real time polymerase chain reaction

DNA was isolated from EDTA-coated whole blood tubes, following patient blood collection, using a TIANamp blood DNA kit (TIANGEN, Beijing, China). The concentration and purity of extracted DNA following the manufacturer’s protocols were determined with a Nanodrop 2000.
Genotyping was performed using TaqMan^® Real Time Polymerase Chain Reaction (PCR) allelic discrimination assays, with a Drug Metabolism Enzyme or predesigned probe and primer (Applied Biosystems, CA, United States), according to the manufacturer’s instructions. TaqMan drug metabolism enzyme genotyping assays (for ABCB1 rs1128503, rs2032582, rs1045642, ABCG2 rs2231142, SLC O 1B1 rs2306283, rs4149056, CYP3A5 rs776746, CYP3A4 rs2242480, ABCC4 rs3742106, rs2274407 and NR1I2 rs1523127) used different PCR conditions from the predesigned TaqMan SNP genotyping assays (for CYP3A4 rs4646437, ABCC4 rs9561778, rs868853, ABCC3 rs4793665, ABCC5 rs562, rs3749438 and NR1I2 rs6785049). Conditions for the former were as follows: 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 90 s. For the latter, 40 cycles of a 1 min annealing/1 min extension were used. Hardy-Weinberg equilibrium tests were performed using chi square tests, where the allele frequencies of the study population were consistent with the law of genetic equilibrium, as indicated by a p-value >0.05.