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

Abstract

BACKGROUND The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies. CONTENT When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored. SUMMARY In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), glutathione synthetase (GSS) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.

Critical appraisal of quantitative PCR results in colorectal cancer research: Can we rely on published qPCR results?

“…Of course, the fact that these six can be assessed for the validity of the reference genes used does not automatically mean they contain reliable results. The results of our analyses are similar to those of a recent study looking at normalisation in the context of the HepaRG cell line, which is widely used as an alternative for primary human hepatocytes (Ceelen et al., 2013). The authors of this study concluded that not one of the 24 reviewed studies used a proper normalization method.…”

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

“…It has been demonstrated that under different experimental conditions and during differentiation, the analysis of HepaRG RT-qPCR data showed a lack of reproducibility and validity as a properly established normalization method had not been applied [13,14]. This was also seen when iPS cells were differentiated to hepatocyte-like cells [15].…”

Reference Gene Validation for RT-qPCR, a Note on Different Available Software Packages

“…Moreover, these guidelines can help researchers with the set-up of qPCR experiments to conform to the posed guidelines and standardize qPCR experimental set-up. Alas, despite a wide acceptance of these guidelines within the specialized field of qPCR, recent literature reviews indicate that these guidelines have not been sufficiently adopted by a larger group of researchers [ 2 , 3 ].…”