Effects of pooling RNA from samples treated with different compounds for determining class specific biomarkers and processes in toxicogenomics

Pooling of RNA samples is generally applied to obtain samples that represent the average signal of biological replicates of a single treatment. For toxicogenomics, pooling RNA of samples treated by different compounds could in the same way summarize these compounds to a single sample with average signals per class. In this study, we investigated the efficiency of such an approach to establish class specific differences in gene expression. As an example we took skin sensitizing compounds as one class and irritating compounds as another. A direct comparison was made to separately hybridized RNA samples. We observed that pooling RNA from compounds of a class substantially increased power to detect significantly regulated genes between classes because variability between pooled samples was much lower. Within pools the vast majority of genes maintained patterns of expression compared to the separately hybridized samples, especially in regulated genes. Both designs yielded appropriate biomarkers. Biomarkers selected from the pooled and separate design performed equally in classification of compounds to their class and relevant processes were found enriched in both designs. Consequently, pooling of RNA of different compound treated samples can be applied to determine class specific biomarkers and processes at much reduced cost and with limited loss of accuracy.

► Pooling RNA of compound treated samples per class is applied to determine behavior of toxicity classes in toxicogenomics. ► Pooling effects are rigorously compared to results from separately hybridized samples. ► A few replicate class pools suffice for biomarker extraction per dose, time, or in vitro system. ► The method can help lowering the threshold towards doing large scale experiments. ► Experimental results are not biased towards one or a few compounds.