Total RNA was extracted from 107 circulating leukocytes (mononuclear cells collected from a 10 ml antecubital venipuncture sample and isolated by ficoll density gradient centrifugation; RNAlater/RNeasy, Qiagen, Valencia, CA, USA), and 5 μg of the resulting RNA was assayed using Affymetrix U133A high-density oligonucleotide arrays [58] in the UCLA DNA Microarray Core as previously described [41,59]. Robust multiarray averaging [60] was applied to quantify expression of the 22,283 assayed transcripts, and differentially expressed genes were identified as those showing ≥30% difference in mean expression levels in samples from high- versus low-lonely individuals (corresponding to a FDR of 10%) [59]. Functional characteristics of individual genes were identified through GO annotations, Gene References into Function annotations, and PubMed literature links retrieved through NCBI Entrez-Gene [61]. Functional commonalities among multiple differentially expressed genes were identified using GOstat [62] with default stringency parameters (Benjamini FDR <0.10) [44]. A full list of differentially expressed genes is provided in Additional data file 1, and raw data are deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO Series GSE7148).
