5 mL of the venous blood was collected into plain (coagulated) and EDTA (anticoaggulated) tubes. Plain vacutainer consist of 3 mL of the serum sample was used to analyze the biochemical parameters and 2 mL of the EDTA sample was used for molecular analysis. Genomic DNA was extracted from peripheral blood leukocytes using Norgen DNA extraction kit (Norgen Biotek corp, Canada). DNA samples were stored at -80°C. The rs1799883 polymorphism was genotyped using a TaqMan® SNP genotyping assay (Assay ID: C_2834835_10) on a 7300HT sequence detection system (Applied Biosystems, USA). Primers and probes were obtained from Applied Biosystems as Assays-by-Design™. Cases and controls were ensured to have even treatment during the assay procedure, and each plate included negative controls (with no DNA). Plates were read on the ABI Prism 7300 using the Sequence Detection Software (Applied Biosystems) using 40 PCR cycles (92°C denaturation for 15 seconds, 60°C annealing/extension for 60 seconds). Measurements were repeated for samples with failed genotypes. Assays that did not show >95% concordance were discarded and replaced with alternative assays with the same tagging properties.
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