For rs12476147 genotyping PCR reactions were performed on a 7900HT real-time sequence detection system using the TaqMan Genotyping Master Mix and the TaqMan SNP genotyping assay (Applied Biosystems). Optimal conditions were as follows: Step 1, 95°C for 10 min; Step 2, 92°C for 15 s, 63°C for 60 s; repeated for 45 cycles. The genotype of each sample was determined by measuring allelic-specific fluorescence using SDS 2.3 software for allelic discrimination (Applied Biosystems). PCR reactions for rs1344706 genotyping were carried out using the LightCycler 480 HRM Master Mix (Roche Applied Science, Indianapolis, IN, USA) and a common forward primer (5′-ggattgggacgaggagaaa-3′) and allele-specific reverse primers (5′-aaacactgaaacaaagaatcaaaaac[T/G]-3′). PCR cycling was performed on a LightCycler 480 (Roche), under the following thermal conditions: Step 1, 95°C for 10 min; Step 2, 95°C for 10 s, 48°C for 15 s, 72°C for 20 s; repeated for 45 cycles. After amplification, samples were kept at 97°C for 1 min and 40°C for 1 min, then melting curves were generated by ramping from 65 to 95°C at 0.02°C/s with continuous fluorescence acquisition. The genotype of each sample was determined by measuring allelic-specific fluorescence using the LightCycler® Software Version 1.5 (Roche).
