Critical selection of reliable reference genes for gene expression study in the HepaRG cell line

The human HepaRG cell line has shown to be a valuable in vitro tool for repeated exposure to chemical compounds and to evaluate their potential toxic outcome. Seen the importance given by the actual EU legislation of cosmetics and chemical substances to the use of in vitro methods in human safety evaluation, one can expect that HepaRG cells will gain importance as human-relevant cell source. At the transcriptional level, RT-qPCR assays are often used to obtain quantitative results. The choice of internal control is important since it may affect the study outcome. Indeed, it is well-known that expression levels of traditional reference genes can vary across tissue types and across experimental settings within one specific tissue type. From a review of the scientific literature, it appears that, for HepaRG cells, S18 often is used as internal control, but without any evidence of its expression stability in this cell line. Therefore, we aimed to select the most optimal reference genes for gene expression studies in HepaRG cells and to check whether S18 is a suitable reference gene. Twelve candidate genes' expression stability level was analyzed by three algorithms (geNorm, BestKeeper, Normfinder), which identified the optimal single reference gene (TBP) and the most suitable set of reference genes (TBP, UBC, SDHA, RLP13, YHWAZ, HMBS, B2M and HPRT1) for HepaRG transcriptional profiling. This study provides a new set of reference genes that is suitable for testing whenever RT-qPCR data for HepaRG cells are generated. The most stable ones can then be selected for further normalization.