Figure 3

Comparison of different normalization strategies. Shown are mRNA expression results from different normalization strategies of TNF-α (panel A, C) and IL10 (panel B, D) in monocytes stimulated for 3 h with LPS compared to non-stimulated cells. The following normalization approaches were applied: The combinations of best-performing reference genes as calculated by geNorm (PPIB, B2M and PPIA; gN comb) or Normfinder (TBP and B2M; NF comb), the best-performing gene as indicated by Normfinder (ACTB) and another frequently used reference gene, GAPDH. (A) and (B) illustrate expression level changes as log2 of the fold change in mRNA levels between stimulated (LPS from N. meningitides, blue bars, and LPS from E. coli, red bars) and non-stimulated cells (not shown). (C) and (D) show the difference in TNF-α and IL10 mRNA, respectively, between monocytes stimulated with equimolar concentrations of LPS from N. meningitides (Nm) and E. coli (Ec) for 3 h based on different normalization approaches. Statistical significance was assessed by Wilcoxon matched pairs signed ranks test, and selected differences are indicated. Results with p < 0.05 are regarded as statistically significant and indicated by asterisks. Paired student's t test was also performed and resulted in similar findings (data not shown). n.s. not significant. Error bars indicate ± SD.