A competitive quantitative polymerase chain reaction assay for bovine transforming growth factor-B1 mRNA

We have developed a flexible reverse transcription (RT) coupled quantitative polymerase chain reaction (PCR) assay for transforming growth factor beta-1 (TGF-b) mRNA. A deletion mutant cDNA internal standard was prepared from the wild type cDNA and used to normalize intersample PCR efficiency differ...

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Bibliographic Details
Published in:Life sciences (1973) 1996, Vol.59 (25-26), p.2157-2165
Main Authors: Lanzillo, J J, Maloney, E K, White, A C, Stevens, J, Fanburg, B
Format: Article
Language:English
Subjects:
Animals
Cattle
Cells, Cultured
DNA, Complementary
Endothelium, Vascular - cytology
Endothelium, Vascular - metabolism
Polymerase Chain Reaction - methods
RNA, Messenger - analysis
Sequence Deletion
Transforming Growth Factor beta - genetics
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Summary:We have developed a flexible reverse transcription (RT) coupled quantitative polymerase chain reaction (PCR) assay for transforming growth factor beta-1 (TGF-b) mRNA. A deletion mutant cDNA internal standard was prepared from the wild type cDNA and used to normalize intersample PCR efficiency differences. The assay is compatible with samples from cow and other species. Using RT-PCR, we determined that TGF-b mRNA in bovine pulmonary artery endothelial cells is increased by TGF-b 7.5-fold within 6h and remains 4-fold above baseline after 12h. In addition, unlike TGF-b bioactivity, mRNA levels in endothelial cells are not decreased upon exposure of the cells to either glutathione (reduced or oxidized), cysteine, or N-acetylcysteine for 24h.
ISSN:0024-3205
DOI:10.1016/S0024-3205(96)00572-3