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BMC molecular biology2008; 9; 49; doi: 10.1186/1471-2199-9-49

Exercise induced stress in horses: selection of the most stable reference genes for quantitative RT-PCR normalization.

Abstract: Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare. In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved in the cellular stress response. Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. Results: The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in geNorm, NormFinder and BestKeeper). Succinate dehydrogenase complex subunit A (SDHA) and hypoxanthine phosphoribosyltransferase (HPRT) always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transferrin receptor (TFRC) and ribosomal protein L32 (RPL32) were constantly classified as the less reliable controls. Conclusions: This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate SDHA and HPRT as the most stable reference genes of our pool.
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Publication Date: 2008-05-19 PubMed ID: 18489742PubMed Central: PMC2412902DOI: 10.1186/1471-2199-9-49Google Scholar: Lookup
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  • 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 identifying the most stable reference genes for normalizing data received from quantitative real-time PCR (qRT-PCR) technology, specifically when studying exercise-induced stress in horses.

Understanding the Study

  • This study is a crucial step in understanding the molecular mechanisms that activate in horses during strenuous exercise and the stress response associated with it. By selecting the most stable reference genes, the researchers can ensure that any changes observed in gene expression are due to the exercise stress and not natural variability or physiological conditions.
  • Microarray and qRT-PCR technologies are often used in research to study gene expression. In qRT-PCR, reference genes act as controls that help calibrate the data, allowing for more accurate interpretations of gene expression changes.
  • An ideal reference gene should have a stable level of expression that is not affected by external parameters such as physiological conditions, the type of experiment being conducted, or individual differences among subjects.

The Evaluation Process

  • The study evaluated the expression of nine potential reference genes in the lymphocytes of ten endurance horses during strenuous exercise. These genes were tested through qRT-PCR.
  • The researchers ranked these genes based on the stability of their expression using three different data analysis methods, namely geNorm, NormFinder, and BestKeeper.
  • The genes succinate dehydrogenase complex subunit A (SDHA) and hypoxanthine phosphoribosyltransferase (HPRT) were consistently ranked as the most stable by all three methods.
  • Genes like glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transferrin receptor (TFRC) and ribosomal protein L32 (RPL32), however, were consistently classified as the most unreliable controls.

Conclusions and Implications

  • This research underlines the importance of carefully selecting reference genes for qRT-PCR studies when evaluating exercise-induced stress in horses.
  • The study concludes that SDHA and HPRT are the most reliable reference genes, based on the stability of their expression across different horses and varying levels of strenuous exercise.
  • Identifying these stable reference genes may improve the accuracy and reliability of future studies using qRT-PCR technology to evaluate molecular stress responses in horses.

Cite This Article

APA
Cappelli K, Felicetti M, Capomaccio S, Spinsanti G, Silvestrelli M, Supplizi AV. (2008). Exercise induced stress in horses: selection of the most stable reference genes for quantitative RT-PCR normalization. BMC Mol Biol, 9, 49. https://doi.org/10.1186/1471-2199-9-49

Publication

BMC molecular biology
ISSN: 1471-2199
NlmUniqueID: 100966983
Country: England
Language: English
Volume: 9
Pages: 49

Researcher Affiliations

Cappelli, Katia
  • Department of Pathology, Diagnostic and Veterinary Clinic, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy. katia.cappelli@unipg.it
Felicetti, Michela
    Capomaccio, Stefano
      Spinsanti, Giacomo
        Silvestrelli, Maurizio
          Supplizi, Andrea Verini

            MeSH Terms

            • Algorithms
            • Animals
            • Electron Transport Complex II / genetics
            • Gene Expression
            • Horse Diseases / diagnosis
            • Horses
            • Hypoxanthine Phosphoribosyltransferase / genetics
            • Lymphocytes / metabolism
            • Physical Conditioning, Animal
            • Polymerase Chain Reaction / methods
            • Reference Standards
            • Stress, Physiological

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            Citations

            This article has been cited 64 times.
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