Figure 3.

Comparison of normalization strategies. A. Venn diagram demonstrating differences in the populations of regulated mRNAs that result when the Ct values from the PCR arrays were not normalized ("None") or normalized against PPIA or GAPDH expression. The analysis is based on 85 of the 96 targets on the PCR arrays which passed an initial quality screen for missing values (see Methods). The intersecting lines define shared and distinct target gene populations. The values refer to the number of genes apparently regulated significantly (fold change ≥ 1.5, p < 0.05) in the time course. The three methods identified the following numbers of significantly regulated genes: no normalization, 62; PPIA normalization, 60; and GAPDH normalization, 73. B. Artifactual down-regulation of HMGB1 mRNA due to normalization against GAPDH. Expression of HMGB1 throughout the time course was determined in the non-normalized (violet line), GAPDH-normalized (black line), and PPIA-normalized (red line) data sets. P values for significance of regulation were determined with one-way ANOVA: no normalization, 0.52; PPIA normalization, 0.074; GAPDH normalization, 0.0037. Fold change with respect to t = 0h is plotted on the y-axis. For each normalization condition, the p value (t test) is indicated at the time point of maximal fold change.