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

Abstract

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.

Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies

“…However, no reference was provided. Moreover, neither of these reference genes were determined to be proper internal controls in HepaRG in our previous experiments (6 ). In one report, no proof of normalization was described at all (see online Supplemental Table 1 for reference).…”

“…RPS18 was used as a single reference gene in almost half of the studies, but in our study it was found to be less valuable or even useless as an internal control in the HepaRG cell line. RPS18 was listed at the bottom of the ranking in geNorm, BestKeeper, and NormFinder, and according to BestKeeper it was not fit at all as a reference gene for use with differentiating and differentiated HepaRG cells (6 ). However, the most critical issue to be argued is that the RPS18 gene was used as an internal control without any evidence of its expression stability in HepaRG in the described experimental design.…”

“…Owing to the growing value and use of the HepaRG cell line, it is essential that the results generated be reliable and biologically relevant. Therefore, we published, in May 2011, the report Critical Selection of Reliable Reference Genes for Gene Expression Study in the HepaRG Cell Line, aiming to improve substantially the reproducibility and validity of RT-qPCR experiments in this in vitro liver-based model (6 ).…”

Normalization of gene expression using SYBR green qPCR: A case for paraoxonase 1 and 2 in Alzheimer's disease brains

“…To our knowledge, only three published papers investigated the expression stability of common ERG to normalize the qPCR expression of genes involved in AD (Gutala and Reddy, 2004;Barrachina et al, 2006;Coulson et al, 2008). Among these reports, only one was performed with the SYBR Green chemistry (Gutala and Reddy, 2004), and none compared the expression stability of their selected ERGs with different mathematical algorithms, as it has been performed in other biological contexts (Ceelen et al, 2011). Therefore, the first purpose of the present work was to identify a robust set of ERGs for mRNA profiling in AD frontal cortex tissue.…”

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

“…For these reasons, it is expected that the use of HepaRG cells will gain global application as a human-relevant cell model. Ceelen et al has presented optimal reference genes for gene expression analyses in HepaRG progenitor cells undergoing differentiation with 2% dimethylsulfoxide (DMSO), where significant phenotypic and molecular changes occurred with time [12]. However, similar studies in fully differentiated HepaRG cells maintained in culture have not yet been published.…”