A new quantitative RT-PCR assay for the vasoconstrictor endothelin

Endothelin has been found to be the most powerful and important factor regulating vasoconstriction in normal and pathological conditions. Rapid degradation and very low concentrations make it difficult to use even very sensitive enzymatic or immunochemical assays for this oligopeptide. We have established a quantitative polymerase chain reaction (PCR) assay for the mRNA of endothelin-l (ET-1), the best characterized of the three endothelin isoforms known so far. Using human umbilical vein endothelial cells as source for endothelin, the absolute number of ET-1 cDNA molecules serving as template for the amplification reaction can be calculated with the aid of an internal standard as competitor for the PCR primers, while relative amounts of ET-1 mRNA are estimated with a new solid-phase enzyme-linked immunosorbent assay for the PCR product of ET-1 and a constitutively expressed single copy gene product, beta-actin. The combination of these supplementary assay systems allows the analysis of endothelin-1 mRNA regulation on a very sensitive level.