Quantification of endothelins, their receptors, and endothelin-converting enzyme mRNAs in rat genitourinary tract using real-time RT-PCR.
BACKGROUND
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 ETA and ETB 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.


CONCLUSIONS
We demonstrated that significant quantitative variations existed in the expression levels of ET-1, ET-3, ECE-1, and ETA and ETB 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 ETA receptor subtype, respectively.
Yono
Makoto
M
Section of Urology Yale University School of Medicine, PO Box 208041, 333 Cedar Street, New Haven, CT 06520, USA.
Takahashi
Wataru
W
Pouresmail
Mehdi
M
Johnson
David R
DR
Foster
Harris E
HE
Weiss
Robert M
RM
Latifpour
Jamshid
J
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis*; D017931:DNA Primers; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000096:biosynthesis*; D066298:In Vitro Techniques; D008297:Male; D008666:Metalloendopeptidases; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011916:Rats, Inbred F344; D017466:Receptors, Endothelin / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014566:Urogenital System / Q000378:metabolism*
D017931:DNA Primers; D016232:Endothelins; D012333:RNA, Messenger; D017466:Receptors, Endothelin; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes