title: Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis.
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.
authors.affiliation: Immunology, University of Manitoba, Winnipeg, MB, Canada.|Children's Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Nour
authors.initials: N
authors.lastname: Eissa
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Hayam
authors.initials: H
authors.lastname: Hussein
authors.affiliation: Immunology, University of Manitoba, Winnipeg, MB, Canada.|Xuanwu Hospital, Capital Medical University, Beijing, China.
authors.forename: Hongxing
authors.initials: H
authors.lastname: Wang
authors.affiliation: Immunology, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Mohammad F
authors.initials: MF
authors.lastname: Rabbi
authors.affiliation: Internal Medicine section of Gastroenterology, University of Manitoba, Winnipeg, MB, Canada.|IBD Clinical and Research Centre, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Charles N
authors.initials: CN
authors.lastname: Bernstein
authors.affiliation: Immunology, University of Manitoba, Winnipeg, MB, Canada.|Children's Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, MB, Canada.|Internal Medicine section of Gastroenterology, University of Manitoba, Winnipeg, MB, Canada.|IBD Clinical and Research Centre, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Jean-Eric
authors.identifier: http://orcid.org/0000-0002-3018-9719
authors.initials: JE
authors.lastname: Ghia
mesh_terms: D000818:Animals; D003092:Colitis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016264:Dextran Sulfate / Q000633:toxicity; D004195:Disease Models, Animal; D015870:Gene Expression; D020869:Gene Expression Profiling; D053583:Interleukin-1beta / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020652:Peptide Elongation Factor 2 / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D035181:TATA-Box Binding Protein / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: C587672:IL1B protein, mouse; D053583:Interleukin-1beta; D020652:Peptide Elongation Factor 2; D012333:RNA, Messenger; D035181:TATA-Box Binding Protein; D014409:Tumor Necrosis Factor-alpha; D016264:Dextran Sulfate