Real Time PCR
Total RNA was isolated from PBMCs using Trizol reagent. RNA (1 μg) was reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) as described by the manufacturer. Real time PCR was performed in duplicate 20 μl reactions containing Platinum SYBR Green qPCR Supermix-UDG (Thermofisher scientific, USA), 150 nM forward and reverse primers, and 2 μl of cDNA. HuPO (human acidic ribosomal protein) primer sequences were obtained from published reports as described previously (27). IFN-γ, TNF-α, IL-6, SOCS1 and SOCS3 sequence specific primers were used. Two‐fold dilutions of cDNA samples were amplified to control amplification efficiency and to determine the optimal concentration required for each primer pair. HuPO was used as a control gene to calculate the ΔCt values for individual samples. The relative amount of cytokine/HuPO transcripts was calculated using the 2-∆∆cT method as described (28). These values were then used to calculate the relative expression of cytokine mRNA in each of the samples tested.