DNA was isolated from blood samples which was done by adding EDTA using the Geneaid ® Blood DNA Mini Kit and deposited at -20°C until the genotyping procedure. Genotyping was performed using the TaqMan ® genotyping assay and Applied Biosystems ® qPCR 7500 Fast Real-Time PCR System to identify rs2796498, rs9803799, and rs2746342. Genotype identification of rs2796498, rs9803799, and rs2746342 was performed using specifically designed TaqMan primer sequences as follows:
The total volume of amplification mixture used in the real-time PCR was 10 mL, including 5 mL TaqMan GTXpress mix, 2.5 mL nucleotide-free water, and a 0.5 mL TaqMan SNP genotyping assay, and 2 mL of genomic DNA sample. The amplicons were set up according to the following program: fourty cycles at hold 95°C for the 20s, at denaturing 95°C for 3s, and then annealing 60°C for 30s.
The total volume of amplification mixture used in the real-time PCR was 10 mL, including 5 mL TaqMan GTXpress mix, 2.5 mL nucleotide-free water, and a 0.5 mL TaqMan SNP genotyping assay, and 2 mL of genomic DNA sample. The amplicons were set up according to the following program: fourty cycles at hold 95°C for the 20s, at denaturing 95°C for 3s, and then annealing 60°C for 30s.