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 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.
Lanzillo
J J
JJ
New England Medical Center, Pulmonary and Critical Care Division, Boston, MA 02111, USA. JLANZILL@opal.tufts.edu
Maloney
E K
EK
White
A C
AC
Stevens
J
J
Fanburg
B
B
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D017384:Sequence Deletion; D016212:Transforming Growth Factor beta / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta