2.3. SNPs Selection and Analysis

We selected 6 potentially functional SNPs of five crucial oxidative stress-related genes, using the public domain of the single nucleotide polymorphism database (dbSNP) at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/snp; accessed on 01 July 2020) and the available literature. We selected SNPs with known genotype distribution in the European population. Our choice was mainly determined by a potential biological significance of the SNPs resulting from their location (detailed description is included in the Discussion section). The most important information about the studied polymorphisms is shown in Table 1.

Basic information of the SOD2, CAT, GPX4, NOS1 and NOS2 polymorphisms.

*—minor allele frequency (MAF) in European population.

Genomic DNA was isolated from peripheral blood collected in tubes containing EDTA, using a SaMag™ System (Sacace Biotecnologies Srl, Vicenza, Como, Italy) and SaMag™ Blood DNA Extraction Kit according to the instrument manufacturer’s instructions. DNA concentrations were determined by the spectrophotometric measurement of absorbance at 260 nm and the purities were calculated by A260/A280 ratio using a Bio-Tek Synergy HT Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA).

The Taq-Man^® SNP Genotyping Assays were performed according to the manufacturer protocol (Life Technologies, Carlsbad, CA, USA). The Taq-Man Assay IDs and thermal cycling conditions for amplifying PCR products are presented in Table 2. All reactions were carried out in a thermal cycler CFX96™ Real- Time PCR Detection System (BIO-RAD, Hercules, CA, USA). The genotypes were determined automatically based on dye-component fluorescent emission data depicted in the X–Y scatter-plot of the CFX Manager ^TM Software (version 3.1).

TaqMan^® SNP Genotyping Assays used in this study.