Optimized PBMC Isolation and RNA Extraction

Collection of blood samples, PBMC isolation, and RNA extraction were carried out according to previously described protocols [25 (link)]. RNA extracts were treated with DNase (Ambion®, California, USA) to remove any possible genomic DNA contaminants, and reverse transcribed using the High Capacity Archive Kit (Applied Biosystems, CA, USA). Maxima SYBR Green/ROX qPCR Master Mix (#K0222) was used for detection of PCR product and mixtures were run on a Thermo Scientific™ PikoReal System (UK). The samples were run in triplicates, and the relative quantification values were calculated using the Δ-Δ ct method relative to the geometric mean of the housekeeping genes GAPDH and ACTB [26 (link)]. Primers sequences are shown in Table 4.