College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.
Alcohol. 2013 Mar;47(2):109-20. doi: 10.1016/j.alcohol.2012.12.003. Epub 2013 Jan 11.
Identification of the transcriptional networks disrupted by prenatal ethanol exposure remains a core requirement to better understanding the molecular mechanisms of alcohol-induced teratogenesis. In this regard, quantitative reverse-transcriptase polymerase chain reaction (qPCR) has emerged as an essential technique in our efforts to characterize alterations in gene expression brought on by exposure to alcohol. However, many publications continue to report the utilization of inappropriate methods of qPCR normalization, and for many in vitro models, no consistent set of empirically tested normalization controls have been identified. In the present study, we sought to identify a group of candidate reference genes for use within studies of alcohol exposed embryonic, placental, and neurosphere stem cells under both conditions maintaining stemness as well as throughout in vitro differentiation. To this end, we surveyed the recent literature and compiled a short list of fourteen candidate genes commonly used as normalization controls in qPCR studies of gene expression. This list included: Actb, B2m, Gapdh, Gusb, H2afz, Hk2, Hmbs, Hprt, Mrpl1, Pgk1, Ppia, Sdha, Tbp, and Ywhaz. From these studies, we find no single candidate gene was consistently refractory to the influence of alcohol nor completely stable throughout in vitro differentiation. Accordingly, we propose normalizing qPCR measurements to the geometric mean C(T) values obtained for three independent reference mRNAs as a reliable method to accurately interpret qPCR data and assess alterations in gene expression within alcohol treated cultures. Highlighting the importance of careful and empirical reference gene selection, the commonly used reference gene Actb was often amongst the least stable candidate genes tested. In fact, it would not serve as a valid normalization control in many cases. Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes driving differentiation, and model the developmental impact of teratogens.
鉴定产前乙醇暴露破坏的转录网络仍然是更好地理解酒精引起的致畸分子机制的核心要求。在这方面，定量逆转录聚合酶链反应（qPCR）已成为我们描述酒精暴露引起的基因表达变化的重要技术。然而，许多出版物继续报告使用不适当的 qPCR 标准化方法，并且对于许多体外模型，尚未确定一组经过验证的经验测试的标准化对照。在本研究中，我们试图确定一组候选参考基因，用于研究维持干性以及整个体外分化过程中暴露于酒精的胚胎、胎盘和神经球干细胞。为此，我们调查了最近的文献，并编制了一份简短的十四种候选基因清单，这些基因通常用作 qPCR 基因表达研究的标准化对照。这包括：Actb、B2m、Gapdh、Gusb、H2afz、Hk2、Hmbs、Hprt、Mrpl1、Pgk1、Ppia、Sdha、Tbp 和 Ywhaz。从这些研究中，我们发现没有一个单一的候选基因始终不受酒精的影响，也没有在整个体外分化过程中完全稳定。因此，我们建议将 qPCR 测量值归一化为三个独立参考 mRNA 的几何平均 C（T）值，作为准确解释 qPCR 数据和评估酒精处理培养物中基因表达变化的可靠方法。强调仔细和经验参考基因选择的重要性，常用的参考基因 Actb 通常是测试中最不稳定的候选基因之一。事实上，在许多情况下，它不能作为有效的归一化对照。这里提供的数据将有助于使用干细胞设计未来的实验，以研究驱动分化的转录过程，并模拟致畸剂对发育的影响。
