Quantitative Real-Time PCR Gene Expression Analysis

RNA from whole blood was extracted with PAXgene® blood RNA kits (Qiagen®, Valencia, CA) as per instructions. Quantity of RNA was determined using a Nanodrop 2000 (ThermoScientific™, Waltham MA). Maxima® first strand cDNA synthesis kit (ThermoScientific™) was used to synthesize cDNA with 2,000 ng of starting RNA. A Beckman BioMek 3000 liquid handling robot was used to set up qPCR reactions into 384 well plates using specific gene primers (27 (link)) (Supplementary Table 2) and 2X perfeCTA™ SYBR® Green SuperMix, ROX™ (Quantabio, Beverly, MA). qPCR reactions were run using an ABI ViiA7 with a holding stage of 3 min at 95°C, followed by 40 cycles of 10 s at 95°C, 30 s at annealing temperature and 30 s at 72°C. Gene expression was calculated using ΔΔCt method with TBP (TATA box binding protein) used as a house keeping gene (28 (link)).