Eight transcripts characteristic of the two key functional GO categories showing differential activity (immune activation and transcriptional control) were selected for independent verification of differential expression by real-time RT-PCR. Results were concordant with microarray indications in seven instances, with RT-PCR confirming over-expression of IL1B,IL8,PTGS2/COX2, FOSB, EGR1, CDC25B, and under-expression of G1P3 (MANOVA difference in all transcripts simultaneously, multivariate p < 0.0001, and p < 0.05 for each transcript analyzed individually; Additional data file 2). Both micorarray data and quantitative RT-PCR found the GR NR3C1 gene to be transcribed at comparable levels in leukocytes from high- versus low-lonely individuals (difference <4% by RT-PCR, p = 0.815, and <15% by microarray, p = 0.112). Expression of CD3 (CD3G, CD3D, CD3E, CD3Z), CD8 (CD8A, CD8B1), CD4, CD56 (NCAM1), CD19, and CD14 were also comparable (all differences <10% in magnitude, all p > 0.20). Thus, observed differences in mononuclear cell gene expression do not stem from variations in the relative prevalence of leukocyte subset mRNAs within the circulating mononuclear cell RNA pool [45-47].
