Immunology, University of Manitoba, Winnipeg, MB, Canada.
Children's Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, MB, Canada.
Sci Rep. 2017 Feb 10;7:42427. doi: 10.1038/srep42427.
2,4-Dinitrobenzene sulfonic acid (DNBS)-induced colitis is an experimental model that mimics Crohn's disease. Appropriateness of reference genes is crucial for RT-qPCR. This is the first study to determine the stability of reference gene expression (RGE) in mice treated with DNBS. DNBS experimental Colitis was induced in male C57BL/6 mice. RNA was extracted from colon tissue and comprehensive analysis of 13 RGE was performed according to predefined criteria. Relative colonic TNF-α and IL-1β mRNA levels were calculated. 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 fluctuation within the inflamed and control groups. Conversely, ribosomal protein large P0 (Rplp0), non-POU domain containing (Nono), TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. TNF-α and IL-1β mRNA levels was dependent on the reference gene used and varied from significant when the most stable genes were used to non-significant when the least stable genes were used. The appropriate choice of RGE is critical to guarantee satisfactory normalization of RT-qPCR data when using DNBS-Model. We recommend using Rplp0, Nono, Tbp, Hprt and Eef2 instead of common reference genes.
2,4-二硝基苯磺酸（DNBS）诱导的结肠炎是一种模拟克罗恩病的实验模型。参考基因的适用性对于 RT-qPCR 至关重要。这是首次研究确定用 DNBS 处理的小鼠中参考基因表达（RGE）的稳定性。在雄性 C57BL/6 小鼠中诱导 DNBS 实验性结肠炎。从结肠组织中提取 RNA，并根据预设标准对 13 个 RGE 进行综合分析。计算结肠 TNF-α和 IL-1β mRNA 水平。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（β2m）在炎症组和对照组内波动最大。相反，核糖体蛋白大 P0（Rplp0）、非 POUM 结构域包含（Nono）、TATA 框结合蛋白（Tbp）和真核翻译延伸因子 2（Eef2）不受炎症影响，是最稳定的基因。TNF-α和 IL-1β mRNA 水平取决于所使用的参考基因，当使用最稳定的基因时，差异具有统计学意义，而当使用最不稳定的基因时，差异无统计学意义。当使用 DNBS 模型时，选择合适的 RGE 对于保证 RT-qPCR 数据的满意归一化至关重要。我们建议使用 Rplp0、Nono、Tbp、Hprt 和 Eef2 代替常用的参考基因。
