Pathlicon nv, Evergem, Belgium;
Clin Chem. 2014 Mar;60(3):451-4. doi: 10.1373/clinchem.2013.209478. Epub 2013 Sep 11.
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.
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.
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.
HepaRG 细胞系被广泛用作多种应用的原代人肝细胞替代物，包括药物筛选，并且作为人类相关细胞来源的重要性日益增加。因此，越来越多的实验正在使用该细胞系进行，包括基因表达研究的实时定量 PCR（RT-qPCR）实验。
当进行 RT-qPCR 实验时，只有注意几个关键方面，包括适当的标准化策略，结果才是可靠的。因此，根据 MIQE（定量实时 PCR 实验发布的最低信息）指南，我们在 2011 年确定了 HepaRG 细胞系统中基因表达研究的最适参考基因。这项研究还提供了明确的证据，表明在 HepaRG 细胞中使用单个参考基因[甘油醛-3-磷酸脱氢酶（GAPDH）、核糖体蛋白 S18（RPS18）或肌动蛋白，β（ACTB）]进行归一化是不够的。我们对我们研究之后发表的相关研究进行筛选后发现，我们的研究结果完全被忽略了。
在我们筛选的 24 项研究中，没有一项使用了适当的归一化方法。在我们筛选的 24 项报告研究中，只有 1 项研究使用了 1 个参考基因进行归一化，其中 RPS18 和 GAPDH 最常用，其次是次黄嘌呤磷酸核糖基转移酶 1（HPRT1）、谷胱甘肽合成酶（GSS）（hGus）、β-2 微球蛋白（B2M）和酸性核糖体磷蛋白 P0（36B4）。有 2 项研究报告了使用多个参考基因（2 个和 3 个），但这些基因在 HepaRG 细胞中的表达稳定性尚未经过预先验证。在 1 项研究中，没有证据表明使用了任何参考基因。目前在 HepaRG 细胞中进行的 RT-qPCR 基因表达研究没有充分考虑或评估参考基因。这样的研究可能会产生错误和与生物学无关的结果。
