Genomic DNA Genotyping Protocol

Genomic DNA was isolated from peripheral blood using the QIAmp DNA Mini Kit (QIAGEN, Germany), following the manufacturer’s instructions. DNA (10 ng) was analysed with NanoDrop and was mixed with Taqman genotyping master mix (Thermo Fisher Scientific, Applied Biosystems, Sweden) and was amplified using the 7500 Fast Real-Time PCR system (Applied Biosystems). Genotypes of SNPs rs2228262 (# C__16170900_10) and rs1478604 (# C___3100547_20) were analysed with the 7500 Fast Real-Time PCR system with allelic discrimination using TaqMan SNP probes (Applied Biosystems) according to a previous protocol [10 (link)]. The two SNPs, belonging to two different haploblocks, one in the 5’UTR and one in one of the exons, were selected from relevant results in the literature. Amplification was performed using an initial cycle at 50 °C for two minutes, followed by one cycle at 95 °C for 10 min, and finally 40 cycles at 95 °C for 15 s and at 60 °C for one minute. Internal controls and negative controls were included in all PCRs. The manual calling option in the allelic discrimination application ABI PRISM 7500 SDS software, version 1.3.1 (Applied Biosystems) was used to assign genotypes. A total call rate of 98.5% was achieved.