title: Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR.
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.
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: Immunology, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: Laëtitia
authors.initials: L
authors.lastname: Kermarrec
authors.affiliation: Large Animal Medicine, William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California Davis, CA, USA.
authors.forename: Hayam
authors.initials: H
authors.lastname: Hussein
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.initials: JE
authors.lastname: Ghia
mesh_terms: D000818:Animals; D015415:Biomarkers; D003092:Colitis / Q000209:etiology* / Q000473:pathology*; D004139:Dinitrofluorobenzene / Q000009:adverse effects / Q000031:analogs & derivatives*; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007413:Intestinal Mucosa / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D020871:RNA Stability; D012333:RNA, Messenger; D060888:Real-Time Polymerase Chain Reaction; D059467:Transcriptome
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger; C045305:2,4-dinitrofluorobenzene sulfonic acid; D004139:Dinitrofluorobenzene