Validation of the 2-ΔCT method for cytokine mRNAs quantification. The ΔCT method for relative quantification requires that the efficiency of targets (cytokines and related molecules) and reference (CypA) amplified in different tubes is approximately the same. Serial dilutions of cDNA were amplified by real-time PCR using gene-specific primers sets. The difference (ΔCT) between cycle threshold (CT) from target and reference was calculated for each dilution. The absolute value of the slope of ΔCT in relation to the logarithm of cDNA concentration should be less than 0.1 . Panel A shows the results of an IL1b/CypA assay where a cDNA preparation was diluted over a 10.000-fold range in three independent experiments. The data were fit using least-squares linear regression analysis (n = 3). Data are means ± SEM. Panel B: The validity of the ΔCT method was controlled for each experiment: using a 10-fold range standard curve using sample pools as calibrators (green diamond) IL1a/CypA, (blue diamond) IL6/CypA, (red diamond) SOCS3/CypA, (purple diamond) TNFa/CypA.