CONFOUNDING FACTORS: In transcriptomics experimentation, confounding factors frequently exist alongside the intended experimental
factors and can severely influence the outcome of a transcriptome analysis. Confounding factors are regularly discussed in
methodological literature, but their actual, practical impact on the outcome and interpretation of transcriptomics experiments
is, to our knowledge, not documented. For instance, in-vivo experimental factors; like Individual, Sample-Composition and
Time-of-Day are potentially formidable confounding factors. To study these confounding factors, we designed an extensive in-vivo
transcriptome experiment (n = 264) with UVR exposure of murine skin containing six consecutive samples from each individual
mouse (n = 64). ANALYSIS APPROACH: Evaluation of the confounding factors: Sample-Composition, Time-of-Day, Handling-Stress,
and Individual-Mouse resulted in the identification of many genes that were affected by them. These genes sometimes showed
over 30-fold expression differences. The most prominent confounding factor was Sample-Composition caused by mouse-dependent
skin composition differences, sampling variation and/or influx/efflux of mobile cells. Although we can only evaluate these
effects for known cell type specifically expressed genes in our complex heterogeneous samples, it is clear that the observed
variations also affect the cumulative expression levels of many other non-cell-type-specific genes.
ANOVA: ANOVA analysis
can only attempt to neutralize the effects of the well-defined confounding factors, such as Individual-Mouse, on the experimental
factors UV-Dose and Recovery-Time. Also, by definition, ANOVA only yields reproducible gene-expression differences, but we
found that these differences were very small compared to the fold changes induced by the confounding factors, questioning
the biological relevance of these ANOVA-detected differences. Furthermore, it turned out that many of the differentially expressed
genes found by ANOVA were also present in the gene clusters associated with the confounding factors.
our overall conclusion is that confounding factors have a major impact on the outcome of in-vivo transcriptomics experiments.
Thus the set-up, analysis, and interpretation of such experiments should be approached with the utmost prudence.