Experimental setting.

A: Work flow of RNA extraction, processing and hybridization at study’s baseline (BL) and follow up (FU) examination. I) Blood collection, monocyte enrichment and RNA isolation were performed following the same standard operating procedures at the study center for both, BL and FU. II) RNA processing including amplification, purification, and dilution was performed using the same protocol at BL and FU, however, these steps were performed by different personnel a different study site. III) At BL, the Illumina HT12 BeadChips version 3 was used for hybridization and at FU the Illumina HT12 BeadChips version 4. At BL, the BeadArray Reader was used for the scan of the beadchips and at FU, the iScan was used. B: Definition of replicated samples. 15 subjects were randomly selected. At BL and FU, RNA was isolated from these 15 subjects. Based on the time point of RNA isolation and the time point of RNA processing, hybridization (preparation) and scan, three groups of sample replicates were defined: i) RNA isolated, prepared and arrays scanned at BL (BL_rep), ii) RNA isolated at BL, stored for 5 years at -80°C, then prepared and arrays scanned at FU (BLFU_rep) and iii) RNA isolated, prepared and scanned at FU (FU_rep). C: Factors affecting observed gene expression differences between replicate measures. BL_rep and BLFU_rep differ by the time point of RNA preparation and array scan and thus reflect technical differences. For BLFU_rep and FU_rep, RNA preparation and array scan were performed at the same time point, therefore, observed variation mainly reflects biological differences. Observed differences between BL_rep and FU_rep comprise technical and biological variation. (rep = replicated sample).