The list of these most variable and the least variable genes across all donors is available as additional file (Additional file 14, Excel work sheet S2). Validation of microarray data by quantitative RT-PCR (qRT-PCR) In order to verify our microarray data we performed qRT-PCR with 14 target genes. IL23A (Interleukin 23 alpha subunit, p19), JUN (Jun oncogene), NALP2 (NLR family, pyrin domain containing 2), FADD (Fas (TNFRSF6)-associated via death domain), SOCS3 (Suppressor of cytokine signaling 3), SOCS5 (Suppressor buy ARRY-438162 of cytokine signaling

5), TLR1 (Toll like receptor 1), SAA (Serum amyloid A2), IL21R (Interleukin 21 receptor), DEFB1 (Defensin beta 1), IL15RA (Interleukin 15 receptor, alpha), PSMB9 (Proteasome subunit beta type 9), IL10 (Interleukin 10) and INHBA

(Inhibin beta A). The relative fold change of target genes was normalized by the relative expression of a pool of 4 reference genes: B2M (Beta SB202190 supplier 2 microglobulin), G6PD (Glucose 6 phosphate dehydrogenase), PGK1 (Phosphoglycerate kinase 1) and SDHA (Succinate dehydrogenase alpha subunit). Normalized fold change for a target gene versus every reference gene was calculated and a mean fold change of these four was the final value. This normalized mean fold change was plotted against the microarray expression fold change for the same target gene and the linear regression showed a correlations coefficient R2 = 0.914 (Additional file 15, Figure S1). IFNγ, IL12A and IL23B expression Since the CodeLink human UniSet I array does not contain a probe for interferon gamma (IFNγ), we additionally performed real time RT-PCR tests with IFNγ specific primers and found the mRNA to be 9.5 fold upregulated by LM, 6.2 fold induced by SA and 1.8 fold induced by SP (Figure

3; Additional file 16, Table S13). We also evaluated the relative expression of IL12A (p35) and L-gulonolactone oxidase IL23B (IL12B) mRNAs. IL12 and IL23 are heterodimeric cytokines, which share the same beta subunit, a protein of 40 KDa (IL12B/IL23B-p40). The combination of p40 with a different alpha subunit forms the physiologically active IL12 (p35p40) or IL23 (p19p40). The IL23B was not found upregulated after statistical evaluation and filtering of the primary microarray data, however IL23A (p19) mRNA was among the most strongly upregulated genes by all three pathogens and hence enhanced expression of the p40 unit was expected. The qRT-PCR data showed clearly that IL23B (IL12B) mRNA expression was increased in the monocytes of all donors. However this upregulation was highly donor-specific and varied between 2 fold and 54 fold for LM infection and reached up to more than 103 fold change for SA (Figure 3; Additional file 16, Table S13). The expression of IL12A (p35) as demonstrated by the qRT-PCR data was regulated at a much lower level with fold change values between +2 and -2 and was also donor specific. Figure 3 Relative quantification of IL12A, IL12B/IL23B, IL23A and IFNγ by real time RT-PCR.