RELATIVE QUANTIFICATION OF ANGIOTENSIN-CONVERTING ENZYME MESSENGER-RNA IN HUMAN SMOOTH-MUSCLE CELLS, MONOCYTES, AND LYMPHOCYTES BY THE POLYMERASE CHAIN-REACTION

A method was developed for relative quantification of angiotensin-conv erting enzyme (ACE) mRNA in as few as 100 cells. After reverse transcr iption of total RNA to cDNA, multiplexed polymerase chain reaction wit h two sets of primers amplified ACE cDNA and that of an internal stand ard glyceraldehyde phosphate dehydrogenase (GAPDH) simultaneously. By adjusting primer pair concentrations, both ACE and GAPDH were amplifie d with constant efficiencies. Macrophage-like U937 histiocytic lymphom a cells ex-pressed ACE mRNA constitutively. Freshly isolated human mon ocytes did not express ACE mRNA initially, but after 4 days in culture had 8% of the amount found in U937 cells. After phorbol ester stimula tion, monocytes transcribed ACE at levels comparable to U937 cells. Hu man smooth muscle cells had ninefold more ACE mRNA than 4-day monocyte s, but 30% less than U937 cells. In contrast, a mixed population of ly mphocytes was devoid Of ACE mRNA. (C) 1994 Academic Press,Inc.