QQ-plots, four best pre-processing combinations (a)–(d). QQ-plots of test statistics of the Mann–Whitney U test, drawn subsamples of the two case groups, 25 replications Loess and quantile normalization performed well in combination with all transformations but no transformation and the VST. The variance stabilizing normalization reached a high total score. Additional file 2: Table S1 provides detailed results for skewness, tail length and the coefficient of variation for all pre-processing methods. Additional file 1: Figure S39 - S75 shows the resulting plots for all 37 pre-processing combinations with a Mean-SD, Bland-Altman and nonparametric Volcano plot. These plots were shown to the blinded readers. Intra-rater reliability was evaluated using the correlation coefficient for the total quality scores which was 0.99 between both rounds.