Figure 4

Influence of different normalization strategies on the estimated target gene expression. The relative change in mRNA expression of IL-2 (A) and FIH (B) in T cells activated by anti-CD3/CD28 microbeads was assessed by applying and comparing different normalization approaches: (i) normalization to the geometric mean of the two most stable genes (RPL13A and IPO8) according to geNorm and NormFinder, (ii) normalization using an unstably expressed single reference gene (HPRT1 or HBB), and (iii) normalization to the geometric mean of a stably (RPL13A) and an unstably (HPRT1 or HBB) expressed gene. Results are expressed as mean fold change (+ SEM) of n = 4 paired samples. Expression changes (stimulated vs. untreated control) were tested for statistical significance using Student's paired t-test and Bonferroni's correction to account for multiple comparisons, and results with p < 0.05 (indicated by asterisks) were considered statistically significant. Different fold changes within the same target are due to different normalization strategies only.