Original Research

Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

Stephen B. Hughes, Melvyn Quan, Alan Guthrie, Martin Schulman
Onderstepoort Journal of Veterinary Research | Vol 80, No 1 | a402 | DOI: https://doi.org/10.4102/ojvr.v80i1.402 | © 2013 Stephen B. Hughes, Melvyn Quan, Alan Guthrie, Martin Schulman | This work is licensed under CC Attribution-NoDerivatives 4.0
Submitted: 03 January 2012 | Published: 30 August 2013

About the author(s)

Stephen B. Hughes, Department of Production Animal Science, University of Pretoria, Onderstepoort, South Africa
Melvyn Quan, Department of Veterinary Tropical Diseases, University of Pretoria, Onderstepoort, South Africa
Alan Guthrie, Equine Research Centre, University of Pretoria, Onderstepoort, South Africa
Martin Schulman, Department of Production Animal Science, University of Pretoria, Onderstepoort, South Africa


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Abstract

The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins) and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation), real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/µLand 891 copies/µL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95%limit of detection), and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor). This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1 receptor physiology in the horse.

Keywords

Equine; Insulin-like growth factor I; Insulin; Receptor; real-time RT-PCR; Development

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