Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis.
Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis.
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DOI：
10.1371/journal.pone.0156289
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Ghia JE
中科院分区：
文献类型：
--
作者：
Eissa N;Hussein H;Wang H;Rabbi MF;Bernstein CN;Ghia JE
已经开发了许多动物模型来表征结肠炎症的复杂性。在葡聚糖硫酸钠（DSS）小鼠实验性结肠炎中，参考基因的选择对于使用定量真实的时间PCR（RT-qPCR）准确定量靶基因至关重要。没有研究涉及参考基因在小鼠DSS实验性结肠炎中的表现。本研究旨在确定DSS-实验性小鼠结肠炎中参考基因表达（RGE）的稳定性。使用5% DSS连续5天诱导雄性C57 BL/6小鼠结肠炎，对照组给予水。从发炎和非发炎结肠提取RNA。使用RT-qPCR，根据预定义的标准对13个RGE进行比较分析，并通过计算阈值循环的差异来确定相对结肠TNF-α和IL-1β基因表达。结肠炎显著改变了粘膜RGE的稳定性。常用的甘油醛-3-磷酸脱氢酶（Gapdh）、β-肌动蛋白（Actb）或β2-微球蛋白（β 2 m）在炎症组和对照组中显示出最高的变异性。相反，TATA盒结合蛋白（Tbp）和真核翻译延伸因子2（Eef 2）不受炎症的影响，是最稳定的基因。结肠TNF-α和IL-1β mRNA水平的正常化依赖于所使用的参考基因。根据用于标准化数据的基因，统计学显著性从使用TBP /Eef 2时的显著性变化到使用Gapdh、Actb或β 2 m时的非显著性。本研究强调了RGE的适当选择，以确保使用该模型时RT-qPCR数据的充分标准化。次优RGE可能解释了已发表研究中有争议的结果。我们建议使用Tbp和Eef 2代替Gapdh、Actb或β 2 m作为参考基因。
Many animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS) experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR). No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE) in DSS-experimental murine colitis. Colitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1β gene expression was determined by calculating the difference in the threshold cycle. Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1β mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or β2m were used. This study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or β2m as reference genes.