Thermal cycler instrument

To assess gene expression analysis, total RNA was isolated from cells and tissues using RiboEx reagent (GeneAll, Seoul, Korea) according to the manufacturer’s protocols. Quality and quantity assessment of total RNAs was performed using a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Complementary DNA (cDNA) synthesis was performed via miRCURY LNA Universal RT cDNA Synthesis Kit (Qiagen, Germantown, MD, USA). Briefly, 2 µL of 5 ng RNA template, 2 µL of 5x miRCURY RT Reaction Buffer, 1 µL of 10x miRCURY RT Enzyme Mix, and 5 µL of RNase-free water were added to the microtubes and incubated at 42 °C for 60 min, followed by 95 °C for 5 min in a thermal cycler instrument (Bio-rad, Hercules, CA, USA). The qRT-PCR was performed to detect the expression level of miRNA using the SYBR green method. Briefly, 5 µL master mix (Exiqon, Vedbæk, Denmark), 0.5 µL primer (4 pmol) (Exiqon) (Table 1), 0.5 µL cDNA template, and 4 µL distilled H₂O (dH₂O) were added to the 0.1 strips (Gunster, New Taipei City, Taiwan). The strips were incubated according to the following thermal cycling protocol: 95 °C for 10 min, 45 cycles, including 95 °C for 10 s, and 60 °C for 60 s using a light cycler system (Roche, Mannheim, Germany).

Genomic DNA was isolated from peripheral blood cells (200 μl whole blood) using a DNA extraction kit (High Pure PCR Template Preparation Kit, Roche, Germany, Cat.No.11796828001) according to manufacturer instructions. The DNA concentration was measured by Nanodrop 2000 (Thermo Fisher, USA). Specific SNPs were genotyped using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) with the PCR reaction performed using super PCR master mix (YEKTA TAJHIZ AZMA, Tehran-Iran, Cat NO: YT1553-YT1554) using a Thermal Cycler instrument (Bio-Rad, CA, USA).
The primer sequences for each PCR reaction is shown in Table 2. The cycle parameters for the PCR analysis were as follows: initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. To identify the miR-146 C/G polymorphism, the PCR product was digested with the restriction enzyme mnlI (Thermo Fisher, USA, REF: ER1071) by incubating the samples at 37°C for 4 h. The miR-155 T/A polymorphism PCR product was incubated at 37°C overnight with the restriction enzyme TSP45I (Thermo Fisher, USA, REF: ER1511) and the digestion products were detected by 3% agarose gel electrophoresis.