Relative quantification of angiotensin-converting enzyme mRNA in human smooth muscle cells, monocytes, and lymphocytes by the polymerase chain reaction.
A method was developed for relative quantification of angiotensin-converting enzyme (ACE) mRNA in as few as 100 cells. After reverse transcription of total RNA to cDNA, multiplexed polymerase chain reaction with two sets of primers amplified ACE cDNA and that of an internal standard glyceraldehyde phosphate dehydrogenase (GAPDH) simultaneously. By adjusting primer pair concentrations, both ACE and GAPDH were amplified with constant efficiencies. Macrophage-like U937 histiocytic lymphoma cells expressed ACE mRNA constitutively. Freshly isolated human monocytes did not express ACE mRNA initially, but after 4 days in culture had 8% of the amount found in U937 cells. After phorbol ester stimulation, monocytes transcribed ACE at levels comparable to U937 cells. Human smooth muscle cells had ninefold more ACE mRNA than 4-day monocytes, but 30% less than U937 cells. In contrast, a mixed population of lymphocytes was devoid of ACE mRNA.
Aschoff
J M
JM
New England Medical Center, Pulmonary Division, Boston, Massachusetts 02111.
Lazarus
D
D
Fanburg
B L
BL
Lanzillo
J J
JJ
D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods; D011651:Pulmonary Artery / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012333:RNA, Messenger; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012194:RNA-Directed DNA Polymerase; D007703:Peptidyl-Dipeptidase A