Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers
  • Colitis / etiology*
  • Colitis / pathology*
  • Dinitrofluorobenzene / adverse effects
  • Dinitrofluorobenzene / analogs & derivatives*
  • Disease Models, Animal
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Intestinal Mucosa / metabolism
  • Intestinal Mucosa / pathology
  • Male
  • Mice
  • RNA Stability
  • RNA, Messenger*
  • Real-Time Polymerase Chain Reaction
  • Transcriptome


  • Biomarkers
  • RNA, Messenger
  • 2,4-dinitrofluorobenzene sulfonic acid
  • Dinitrofluorobenzene

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