Quantification of endothelins, their receptors, and endothelin-converting enzyme mRNA in rat genitourinary tract using real-time RT-PCR

Abstract

Introduction: Quantification of mRNA expression is essential for the assessment of endothelin (ET) receptor-mediated mechanisms. Recently, a novel technique for the determination of mRNA expression, termed real-time reverse transcription polymerase chain reaction (RT-PCR), has been developed. We therefore applied real-time PCR using SYBR Green I to quantify ET-1, ET-3, ET-converting enzyme-1 (ECE-1), and ET A and ET B receptor subtype mRNA expression in the rat genitourinary tract. Methods: The cDNA was synthesized by RT of RNA extracted from the rat bladder, ventral prostate, dorsolateral prostate, and vas deferens. All steps subsequent to the RT reaction were carried out by the thermal cycler/detector and computer-assisted programs processed a quantitative result. Results: Designing optimal primer sequences that minimized primer-dimer formation and adjusting annealing temperatures that prevented nonspecific product amplification have made it possible to identify a single peak in the melt curve and to obtain an appropriate standard curve for each gene transcript. In our experiments, input cDNA levels as low as 100 copies of the product could be detected. Discussion: We demonstrated that significant quantitative variations existed in the expression levels of ET-1, ET-3, ECE-1, and ET A and ET B receptor subtype mRNAs within a tissue and between different regions of the genitourinary tract and that the predominant expression of ETs and their receptor mRNAs in all tissues studied were ET-1 and the ET A receptor subtype, respectively.