Whole blood was taken from the subjects and genomic DNA was extracted using isolation kit (DNGTM—Plus; SinaClon, Iran) according to the manufacturer's protocol. Genetic SNPs of IL-1α (rs1800587), IL-1β (rs1143627, rs16944, rs1143634), IL-8 (rs4073), and TNF-α (rs1799964, rs1800630, rs1799724, and rs361525) genes were analyzed using primers and respective restriction endonucleases by PCR-based restricted fragment length polymorphism (PCR-RFLP) as described previously (20 (link)–25 (link)).
IL-1α, IL-1β, IL-8, and TNF-α gene expression was evaluated by quantitative RT-PCR, based on RNA extraction and cDNA synthesis from blood leucocytes of all subjects using RNX-Plus kit (SinaClon, Iran) according to the manufacturer's instructions. Expression of candidate genes, as well as housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, was analyzed by real-time RT-PCR using SYBR Green assays. Primer sequences and conditions were designed by ABI PCR equipment (Applied Biosystems, USA) except GAPDH which obtained from previously published sequences (26 (link)) (Supplementary Table 1 ).
IL-1α, IL-1β, IL-8, and TNF-α gene expression was evaluated by quantitative RT-PCR, based on RNA extraction and cDNA synthesis from blood leucocytes of all subjects using RNX-Plus kit (SinaClon, Iran) according to the manufacturer's instructions. Expression of candidate genes, as well as housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, was analyzed by real-time RT-PCR using SYBR Green assays. Primer sequences and conditions were designed by ABI PCR equipment (Applied Biosystems, USA) except GAPDH which obtained from previously published sequences (26 (link)) (