Venous blood was collected between 1100 and 1200 h in PAXgene tubes (Qiagen, Valencia) from each subject and was incubated at room temperature for 24 h for RNA stabilization. RNA was extracted from whole blood according to the manufacturer’s guidelines by using the PAXgene Blood RNA System Kit (PreAnalytix GmbH, Hombrechtikon, Switzerland). The RNA was quantified by optical density readings at A260 nm by using the NanoDrop ND-1000 (Thermo Scientific, Rockford). Gene expression analysis was performed using Agilent Human Genome 4×44 K arrays (Agilent Technologies, Santa Clara). Raw signal data for each of the 3 independent sample groups were analyzed separately by the GeneSpring GX software (Agilent Technologies). Data were filtered according to the expression level for quality control to eliminate genes that were below the 20th percentile threshold. The expression value of each gene was normalized to the median expression value of all genes in each chip. A total of 30,465 probes were included in the analysis.
