Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies
Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies
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DOI：
10.1373/clinchem.2013.209478
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发表时间：
2014-03-01
影响因子：
9.3
通讯作者：
De Spiegelaere, Ward
中科院分区：
文献类型：
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作者：
Ceelen, Liesbeth;De Craene, Jurgen;De Spiegelaere, Ward
背景：HepaRG细胞系被广泛用作原代人类肝细胞的替代品，用于药物筛选等多种应用，并逐渐成为与人类相关的细胞来源。因此，越来越多的实验正在对这种细胞系进行，包括用于基因表达研究的实时定量聚合酶链式反应(RT-qPCR)实验。内容：当进行RT-qPCR实验时，只有注意几个关键方面，包括适当的归一化策略，结果才是可靠的。因此，2011年，我们根据MIQE(发布定量实时聚合酶链式反应实验的最小信息)指南，确定了在HepaRG细胞系统中进行基因表达研究的最佳参考基因。这项研究还提供了明确的证据，表明单一参考基因[甘油醛-3-磷酸脱氢酶(GAPDH)、核糖体蛋白S18(RPS18)或肌动蛋白，β(ACTB)]的使用不足以使HepaRG细胞正常化。我们对研究后发表的相关研究的筛选表明，我们的研究结果被完全忽视。总结：在24项回顾研究中，没有一项使用了适当的归一化方法。在我们筛选的24项研究中，有21项只有1个参考基因被纳入标准化，其中使用频率最高的是RPS18和GAPDH，其次是次黄嘌呤磷酸核糖转移酶1(HPRT1)、谷胱甘肽合成酶(GSS)(HGUS)、β-2微球蛋白(B2M)和酸性核糖体磷酸蛋白P0(36B4)。在两项研究中，报告了多个参考基因(2和3)的使用，但这些基因在HepaRG细胞中的表达稳定性尚未得到预先验证。在一项研究中，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的、与生物学无关的结果。(C)2013年美国临床化学协会
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), beta-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. (C) 2013 American Association for Clinical Chemistry