Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis

PLoS One. 2016 May 31;11(5):e0156289. doi: 10.1371/journal.pone.0156289. eCollection 2016.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

MeSH terms

  • Animals
  • Colitis / genetics*
  • Colitis / metabolism
  • Colitis / pathology
  • Dextran Sulfate / toxicity
  • Disease Models, Animal
  • Gene Expression
  • Gene Expression Profiling
  • Interleukin-1beta / genetics
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Peptide Elongation Factor 2 / genetics
  • RNA Stability
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction
  • TATA-Box Binding Protein / genetics
  • Tumor Necrosis Factor-alpha / genetics

Substances

  • IL1B protein, mouse
  • Interleukin-1beta
  • Peptide Elongation Factor 2
  • RNA, Messenger
  • TATA-Box Binding Protein
  • Tumor Necrosis Factor-alpha
  • Dextran Sulfate

Grant support

This study was supported by grants from Canadian Foundation for Innovation, Crohn’s and Colitis Canada, Research Manitoba, the Canadian Institutes of Health Research to JEG and the Children's Hospital Research Institute of Manitoba to NE and JEG.