A COMPETITIVE QUANTITATIVE POLYMERASE CHAIN-REACTION ASSAY FOR BOVINETRANSFORMING GROWTH FACTOR-B-1, MESSENGER-RNA

We have developed a flexible reverse transcription (RT) coupled quanti tative polymerase chain reaction (PCR) assay for transforming growth f actor beta-1 (TGF-b) mRNA. A deletion mutant cDNA internal standard wa s prepared from the wild type cDNA and used to normalize intersample P CR efficiency differences. The assay is compatible with samples from c ow and other species. Using RT-PCR, we determined that TGF-b mRNA in b ovine pulmonary artery endothelial cells is increased by TGF-b 7.5-fol d within 6h and remains 4-fold above baseline after 12h. In addition, unlike TGF-b bioactivity, mRNA levels in endothelial cells are not dec reased upon exposure of the cells to either glutathione (reduced or ox idized), cysteine, or N-acetylcysteine for 24h.