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.