Department of Internal Medicine, Division of Endocrinology University of Cincinnati College of Medicine, Cincinnati, OH, USA.
Int J Obes (Lond). 2014 Feb;38(2):192-7. doi: 10.1038/ijo.2013.86. Epub 2013 May 24.
Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular end points. Real-time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to have been overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs re-fed and chow vs high-fat diet (HFD) conditions.
Male long-Evans rats were treated under four conditions: chow/fasted, chow/re-fed, HFD/fasted and HFD/re-fed. Expression stabilities of 13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum and ileum using the ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm.
Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. B2M and RPLP0 in the hypothalamus, RPS18 and HMBS in the duodenum, RPLP2 and RPLP0 in the jejunum and RPS18 and YWHAZ in the ileum were the most suitable pairs for a normalization study when the four aforementioned experimental conditions were considered.
Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues have an integral role in the regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression studies.
肥胖具有复杂的代谢病理学，要定义肥胖的潜在机制需要进行具有分子终点的综合研究。实时定量 PCR（RT-qPCR）是一种广泛应用的强大工具。然而，在肥胖相关研究中，似乎忽视了在 RT-qPCR 中使用经过精心验证的参考基因的重要性。本研究的目的是选择一组表达稳定的参考基因，用于在禁食与再喂养以及标准饮食与高脂肪饮食（HFD）条件下的大鼠 RT-qPCR 标准化。
雄性长爪沙鼠接受以下四种处理：标准饮食/禁食、标准饮食/再喂养、高脂肪饮食/禁食和高脂肪饮食/再喂养。使用 ReFinder 软件程序评估候选 13 个参考基因在大鼠下丘脑、十二指肠、空肠和回肠中的表达稳定性。使用 geNorm 确定 RT-qPCR 分析所需的最佳参考基因数量。
使用 geNorm 分析，我们发现，在特定实验条件下，对于每种组织，使用两个最稳定表达的基因作为 RT-qPCR 分析的参考就足够了。当考虑上述四种实验条件时，B2M 和 RPLP0 是下丘脑的最佳组合，RPS18 和 HMBS 是十二指肠的最佳组合，RPLP2 和 RPLP0 是空肠的最佳组合，而 RPS18 和 YWHAZ 是回肠的最佳组合。
我们的研究表明，在急性或慢性能量状态变化时，肥胖相关研究中常用的参考基因（如 ACTB 或 RPS18）的基因表达水平会发生改变。这些发现强调了在研究大鼠下丘脑和肠道时，使用在实验条件下表达稳定的参考基因的重要性，因为这些组织在调节能量平衡方面发挥着重要作用。我们希望这项研究能够引起肥胖研究人员对在基因表达研究中验证参考基因的必要性的认识。
