Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor-1 receptor and insulin receptor expression in equine tissue

Abstract

has been significant progress in the development of new technologies and methodologies to characterize gene expression. The fluorescent-based real-time reverse transcription (RT) polymerase chain reaction (PCR) is an important tool used for clinical and molecular research, biotechnology and as a diagnostic test. Insulin-like growth factors (IGF-1 and IGF-2) and insulin are ubiquitously expressed and play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. The IGF system (IGF-1, IGF-2, IGF -1 receptor, IGF-2 receptor and six IGF-binding proteins) and insulin are consequently essential to most aspects of male and female reproduction. IGF-1 is produced in multiple tissues but predominately in the liver, from where it enters the circulation. Insulin is secreted by β-cells of the pancreas’ islets of Langerhans. Both IGF-1 and insulin polypeptides bind to specific cell surface receptors. These receptors are members of the superfamily known as tyrosine protein kinases, and are composed of two α and two β subunits linked by disulfide bonds to form an αβ–αβ heterotetramer. The α subunits include ligand binding sites, whereas the β subunits contain tyrosine kinase activity. The aim of this project was to develop real-time RT-PCR assays for quantification of equine insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (INS-R) mRNA. The assays were developed using stallion testicular tissue samples, obtained by excisional biopsy, from three horse breeds (Friesan, Thoroughbred and Warmblood). The assays developed were efficient, sensitive and had a broad linear range of detection (seven logs for IGF-1R and six logs for INS-R). The assays worked well in our hands and were both sensitive and specific for the detection of equine IGF-1R and INS-R mRNA in a variety of equine tissues.