OpenLB
Open Library of Bioscience
Advanced Search
PloS one
2016;11 (5): e0156289.

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

Nour Eissa, Hayam Hussein, Hongxing Wang, Mohammad F Rabbi, Charles N Bernstein, Jean-Eric Ghia
Author Information
  1. Nour Eissa: Immunology, University of Manitoba, Winnipeg, MB, Canada.
  2. Hayam Hussein: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Ohio State University, Columbus, Ohio, United States of America.
  3. Hongxing Wang: Immunology, University of Manitoba, Winnipeg, MB, Canada.
  4. Mohammad F Rabbi: Immunology, University of Manitoba, Winnipeg, MB, Canada.
  5. Charles N Bernstein: Internal Medicine section of Gastroenterology, University of Manitoba, Winnipeg, MB, Canada.
  6. Jean-Eric Ghia: Immunology, University of Manitoba, Winnipeg, MB, Canada. ORCID
PMID: 27244258 DOI: 10.1371/journal.pone.0156289

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.

References

  1. J Biotechnol. 1999 Oct 8;75(2-3):291-5 [PMID: 10617337]
  2. Genome Biol. 2002 Jun 18;3(7):RESEARCH0034 [PMID: 12184808]
  3. EMBO J. 2004 Mar 10;23(5):1008-19 [PMID: 14976550]
  4. Biotechnol Lett. 2004 Mar;26(6):509-15 [PMID: 15127793]
  5. Cancer Res. 2004 Aug 1;64(15):5245-50 [PMID: 15289330]
  6. Nat Methods. 2005 Oct;2(10):731-4 [PMID: 16179916]
  7. BMC Genomics. 2005 Nov 02;6:150 [PMID: 16266432]
  8. Am J Physiol Gastrointest Liver Physiol. 2006 May;290(5):G1067-74 [PMID: 16399877]
  9. Physiol Genomics. 2006 Apr 13;25(2):242-9 [PMID: 16464976]
  10. J Neurosci Methods. 2006 Sep 30;156(1-2):101-10 [PMID: 16554095]
  11. Gastroenterology. 1990 Mar;98(3):694-702 [PMID: 1688816]
  12. J Immunol. 2006 Sep 1;177(5):3273-82 [PMID: 16920968]
  13. BMC Mol Biol. 2006 Oct 06;7:33 [PMID: 17026756]
  14. BMC Genomics. 2007 May 22;8:127 [PMID: 17519037]
  15. PLoS One. 2007 Sep 19;2(9):e898 [PMID: 17878933]
  16. BMC Mol Biol. 2008 May 06;9:46 [PMID: 18460208]
  17. Nat Protoc. 2008;3(6):1101-8 [PMID: 18546601]
  18. Biochem Biophys Res Commun. 2008 Sep 12;374(1):106-10 [PMID: 18602371]
  19. Neuroscience. 2008 Oct 2;156(2):305-9 [PMID: 18722514]
  20. Clin Chem. 2009 Apr;55(4):611-22 [PMID: 19246619]
  21. Nucleic Acids Res. 2009 Nov;37(21):e138 [PMID: 19734345]
  22. Sex Dev. 2009;3(4):194-204 [PMID: 19752599]
  23. Anal Biochem. 2010 Apr 15;399(2):211-7 [PMID: 20036209]
  24. Mol Vis. 2010 Jun 11;16:1076-86 [PMID: 20596249]
  25. BMC Mol Biol. 2010 Aug 16;11:60 [PMID: 20712867]
  26. Inflamm Bowel Dis. 2011 Jul;17(7):1603-9 [PMID: 21053357]
  27. Gastroenterology. 2011 May;140(6):1720-28 [PMID: 21530738]
  28. BMC Res Notes. 2011 Oct 14;4:410 [PMID: 21996334]
  29. Gastroenterology. 2012 Jan;142(1):46-54.e42; quiz e30 [PMID: 22001864]
  30. PLoS One. 2011;6(11):e26980 [PMID: 22087239]
  31. Science. 1990 Jun 29;248(4963):1625-30 [PMID: 2363050]
  32. J Biomol Screen. 2013 Oct;18(9):1008-17 [PMID: 23690294]
  33. Inflamm Bowel Dis. 2013 Dec;19(13):2840-7 [PMID: 24141710]
  34. Curr Protoc Immunol. 2014 Feb 04;104:Unit 15.25. [PMID: 24510619]
  35. Biochem Pharmacol. 2014 Jun 1;89(3):386-98 [PMID: 24637240]
  36. PLoS One. 2015 Jan 24;10(1):e0117058 [PMID: 25617865]
  37. Genes Cells. 2015 Aug;20(8):625-35 [PMID: 26059597]
  38. PLoS One. 2015 Nov 25;10(11):e0142536 [PMID: 26605545]
  39. J Basic Microbiol. 2016 Sep;56(9):986-98 [PMID: 27112251]
  40. Lab Invest. 1993 Aug;69(2):238-49 [PMID: 8350599]

MeSH Term

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

Chemicals

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

Word Cloud

Similar Articles

Cited By