FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Vet Immunol Immunopathol / Determination of internal control for gene expression studie...
Veterinary immunology and immunopathology2009; 130(1-2); 114-119; doi: 10.1016/j.vetimm.2009.01.012

Determination of internal control for gene expression studies in equine tissues and cell culture using quantitative RT-PCR.

Abstract: Quantitative reverse transcription polymerase chain reaction (RT-PCR) has become a basic, reliable and sensitive modern technique, in both biological research and clinical diagnosis, for investigation of gene expression and validation of cDNA microarray analysis. Accurate mRNA quantification using quantitative RT-PCR commonly requires data normalization through stable housekeeping genes (HKGs). Selection of HKGs for data normalization is critical for accurate mRNA quantification. Our objective was to evaluate a set of candidate HKGs as internal controls for gene expression studies using quantitative RT-PCR in equine tissues and cell culture. One-step quantitative RT-PCR for 6 HKGs was performed using total RNA from equine tissue samples and cultured peripheral blood mononuclear cells (PBMCs). The stability of HKGs was mainly evaluated by analysis of variance, analyses of the standard deviation and coefficient of variation of Ct, and change of Ct of HKGs between control and treated samples. 18S rRNA consistently showed the smallest standard deviation and coefficient of variation, and the least change of Ct between control and treated samples, thus was identified as the most stable HKG for mRNA data normalization in quantitative RT-PCR for studying gene expression in equine tissues and cultured PBMCs.
Get Full Text
Publication Date: 2009-02-04 PubMed ID: 19269038DOI: 10.1016/j.vetimm.2009.01.012Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research article focuses on finding the most reliable internal controls for gene expression studies in equine tissues and cell cultures through the use of quantitative reverse transcription polymerase chain reaction (RT-PCR). The study identifies 18S rRNA as the most stable housekeeping gene, thus making it suitable for data normalization in this context.

Objective and Methodology

  • The research aimed to identify a suitable set of housekeeping genes (HKGs) as internal controls for accurate mRNA quantification in equine tissues and cell culture gene expression studies using RT-PCR.
  • The candidate HKGs were evaluated through a one-step quantitative RT-PCR process, applied to total RNA extracted from equine tissue samples and cultured peripheral blood mononuclear cells (PBMCs).

Evaluation of Housekeeping Genes

  • Housekeeping genes considered for the study were evaluated based on several criteria such as their stability—which was assessed through analysis of variance, standard deviation analyses, coefficient of variation of Ct, and change of Ct of HKGs between control and treated samples.
  • The selection process was determined by identifying the gene that showed minimum changes, relating to these measures, between the control and treated samples.

Study Findings

  • The study revealed that 18S rRNA was the most stable HKG, as it demonstrated the smallest standard deviation and coefficient of variation, and the least change of Ct between control and treated samples.
  • Based on these results, the study concluded that 18S rRNA is the most suitable for mRNA normalization during quantitative RT-PCR for studying gene expression in equine tissues and cultured PBMCs.

Study Implications and Significance

  • The accurate selection of housekeeping genes as internal controls is crucial for precise mRNA quantification, as this affects the validity of gene expression studies. Incorrect normalization can lead to misinterpretation of results.
  • By identifying 18S rRNA as a reliable HKG in studies using equine tissues and cultured PBMCs, the study contributes to enhancing the accuracy of gene expression analysis in these contexts.
  • Such findings may play a crucial role in both biological research and clinical diagnostic processes, improving their overall reliability and validity.

Cite This Article

APA
Zhang YW, Davis EG, Bai J. (2009). Determination of internal control for gene expression studies in equine tissues and cell culture using quantitative RT-PCR. Vet Immunol Immunopathol, 130(1-2), 114-119. https://doi.org/10.1016/j.vetimm.2009.01.012

Publication

Veterinary immunology and immunopathology
ISSN: 1873-2534
NlmUniqueID: 8002006
Country: Netherlands
Language: English
Volume: 130
Issue: 1-2
Pages: 114-119

Researcher Affiliations

Zhang, Yuwen W
  • Department of Anatomy and Physiology, Kansas State University, Manhattan, KS, USA; Department of Clinical Sciences, Kansas State University, Manhattan, KS 66506, USA.
Davis, Elizabeth G
    Bai, Jianfa

      MeSH Terms

      • Animals
      • Gene Expression Profiling / methods
      • Gene Expression Profiling / veterinary
      • Horses / genetics
      • RNA, Messenger / chemistry
      • RNA, Messenger / genetics
      • RNA, Ribosomal, 18S / chemistry
      • RNA, Ribosomal, 18S / genetics
      • Reverse Transcriptase Polymerase Chain Reaction / methods
      • Reverse Transcriptase Polymerase Chain Reaction / veterinary

      Citations

      This article has been cited 13 times.
      1. Riveroll AL, Skyba-Lewin S, Lynn KD, Mubyeyi G, Abd-El-Aziz A, Kibenge FST, Kibenge MJT, Cohen AM, Esparza-Gonsalez B, McD￮ L, Montelpare WJ. Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR.. Genes (Basel) 2023 Mar 8;14(3).
        doi: 10.3390/genes14030673pubmed: 36980946google scholar: lookup
      2. Seo MG, Ouh IO, Kwak D. Detection and Genotypic Analysis of Anaplasma bovis and A. phagocytophilum in Horse Blood and Lung Tissue.. Int J Mol Sci 2023 Feb 7;24(4).
        doi: 10.3390/ijms24043239pubmed: 36834651google scholar: lookup
      3. Nino-Fong R, Esparza Gonzalez BP, Rodriguez-Lecompte JC, Montelpare W, McD￮ L. Development of a biologically immortalized equine stem cell line.. Can J Vet Res 2021 Oct;85(4):293-301.
        pubmed: 34602734
      4. Fagre AC, Lewis J, Miller MR, Mossel EC, Lutwama JJ, Nyakarahuka L, Nakayiki T, Kityo R, Nalikka B, Towner JS, Amman BR, Sealy TK, Foy B, Schountz T, Anderson J, Kading RC. Subgenomic flavivirus RNA (sfRNA) associated with Asian lineage Zika virus identified in three species of Ugandan bats (family Pteropodidae).. Sci Rep 2021 Apr 16;11(1):8370.
        doi: 10.1038/s41598-021-87816-5pubmed: 33863991google scholar: lookup
      5. Borkowska P, Zielińska A, Paul-Samojedny M, Stojko R, Kowalski J. Evaluation of reference genes for quantitative real-time PCR in Wharton's Jelly-derived mesenchymal stem cells after lentiviral transduction and differentiation.. Mol Biol Rep 2020 Feb;47(2):1107-1115.
        doi: 10.1007/s11033-019-05207-6pubmed: 31781918google scholar: lookup
      6. Yan L, Toohey-Kurth KL, Crossley BM, Bai J, Glaser AL, Tallmadge RL, Goodman LB. Inhibition monitoring in veterinary molecular testing.. J Vet Diagn Invest 2020 Nov;32(6):758-766.
        doi: 10.1177/1040638719889315pubmed: 31735123google scholar: lookup
      7. Stasiak IM, Smith DA, Ganz T, Crawshaw GJ, Hammermueller JD, Bienzle D, Lillie BN. Iron storage disease (hemochromatosis) and hepcidin response to iron load in two species of pteropodid fruit bats relative to the common vampire bat.. J Comp Physiol B 2018 Jul;188(4):683-694.
        doi: 10.1007/s00360-018-1155-4pubmed: 29594459google scholar: lookup
      8. Azarpeykan S, Dittmer KE. Evaluation of housekeeping genes for quantitative gene expression analysis in the equine kidney.. J Equine Sci 2016;27(4):165-168.
        doi: 10.1294/jes.27.165pubmed: 27974876google scholar: lookup
      9. Nazari F, Parham A, Maleki AF. GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells.. J Anim Sci Technol 2015;57:18.
        doi: 10.1186/s40781-015-0050-8pubmed: 26290738google scholar: lookup
      10. Finno CJ, Stevens C, Young A, Affolter V, Joshi NA, Ramsay S, Bannasch DL. SERPINB11 frameshift variant associated with novel hoof specific phenotype in Connemara ponies.. PLoS Genet 2015 Apr;11(4):e1005122.
        doi: 10.1371/journal.pgen.1005122pubmed: 25875171google scholar: lookup
      11. Cadby JA, Buehler E, Godbout C, van Weeren PR, Snedeker JG. Differences between the cell populations from the peritenon and the tendon core with regard to their potential implication in tendon repair.. PLoS One 2014;9(3):e92474.
        doi: 10.1371/journal.pone.0092474pubmed: 24651449google scholar: lookup
      12. Karagianni AE, Kapetanovic R, McGorum BC, Hume DA, Pirie SR. The equine alveolar macrophage: functional and phenotypic comparisons with peritoneal macrophages.. Vet Immunol Immunopathol 2013 Oct 1;155(4):219-28.
        doi: 10.1016/j.vetimm.2013.07.003pubmed: 23978307google scholar: lookup
      13. Beekman L, Tohver T, Dardari R, Léguillette R. Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease.. BMC Mol Biol 2011 Jan 28;12:5.
        doi: 10.1186/1471-2199-12-5pubmed: 21272375google scholar: lookup
      Subscribe to our newsletter
      Join 99,383 horse owners like you who receive our email updates on horse health and nutrition.