Abstract
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.
Highlights
In uneven exposure to DSS, in which causes variation in the degree, extent, and distribution of mucosal damage in the colon[4]
Induction and development of experimental colitis was confirmed in the dinitrobenzene sulfonic acid (DNBS)-treated group compared to controls
We evaluated the suitability of 13 genes, including the most commonly-used reference genes Gapdh, Actb, Rplp[0], β2m, and Hprt, which have been used in normalizing messenger RNA (mRNA) expression in normal and pathological intestinal mucosa[33,34,35]
Summary
In uneven exposure to DSS, in which causes variation in the degree, extent, and distribution of mucosal damage in the colon[4]. Evaluation of 13 potential reference genes (Gapdh, Actb, β2m, Hmbs, Hprt, RpLp0, Tbp, Gusb, Ppia, Oaz[1], Nono, Tfrc, and Eef2) was assessed in colitic mice by analyzing reference gene stability using different algorithms such as geNorm[23], bestKeeper[24], normFinder[25], the comparative delta Ct method[26], and the comprehensive ranking[27] These approaches provided a detailed comprehensive analysis of common and novel reference genes for the use of mouse DNBS-induced colitis in relation to RT-qPCR experiments. To validate the influence of reference gene stability on target gene normalization, the relative gene expression of two major colonic pro-inflammatory cytokines TNF-α and IL-1β in inflamed and non-inflamed colonic tissues was evaluated using the 13 housekeeping genes to find the most suitable endogenous normalizer genes for mRNA expression
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