A competitive deletion mutant quantitative PCR assay for angiotensin-converting enzyme mRNA in smooth muscle cells.
To quantify angiotensin-converting enzyme (ACE) mRNA, we have developed a reverse transcription (RT)-coupled competitive PCR (RT-PCR) assay with a deletion mutant internal standard. The RT-PCR detects ACE mRNA from both human and bovine sources. ACE mRNA was detected in total RNA from cultured human saphenous vein smooth muscle cells (HuSV-SMCs) and from bovine pulmonary artery (BPA) SMCs. BPA-SMC expressed ninefold less ACE mRNA than BPA endothelial cells, and threefold less than HuSV-SMCs. Apparent amounts of ACE mRNA were 118,350 +/- 2,300 copies in HuSV-SMCs and 42,200 +/- 11,300 copies in BPA-SMCs per microgram of total cell RNA. The accuracy of the absolute values is subject to the limitations of the assumptions used to calculate them. These data support the hypothesis that components of the renin-angiotensin system are transcribed by SMCs.
Lanzillo
J J
JJ
New England Medical Center, Pulmonary and Critical Care Division, Boston, Massachusetts 02111, USA.
Kong
X J
XJ
Fanburg
B L
BL
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D016296:Mutagenesis; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods; D011651:Pulmonary Artery / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase; D012501:Saphenous Vein / Q000201:enzymology; D017384:Sequence Deletion
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D007703:Peptidyl-Dipeptidase A