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667 QUANTITATIVE EXPRESSION OF ACAT-1 AND ACAT-2 GENES IN HUMAN LIVER AND DUODENUM BY REAL-TIME PCR K. Rangaray1, R. Simpson1, D.J. MacLean1, L.K. Nathanson2, K.A. Stuart3, S.P. Scott4, J. De Jersey1, J. Smith1,2 1 Department of Biochemistry and Molecular Biology, The University of Queensland, 2Department of Surgery, The University of Queensland, 3 Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, 4Queensland Institute of Medical Research, Brisbane, Australia Acyl-CoA:cholesterol acyltransferase (ACAT) catalyses the esterification of cholesterol with long chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis (e.g. cholesterol adsorption, VLDL secretion and cholesterol ester formation in atheroma), making it an obvious target for pharmacological inhibition. Two ACAT genes termed ACAT-1 and ACAT-2 are known. Although these genes appear to display differential tissue expression, assay procedures to quantify abundance of their mRNA transcripts or protein products have not been described. In this study we have developed a quantitative multiplex assay using TaqManTM Real-Time PCR technology to measure the relative abundance of ACAT-1 and ACAT- 2 transcripts in human liver and duodenum. RNA samples were isolated from liver from organ donors and duodenal biopsies from patients having gastroscopy for clinically indicated reasons. mRNA representing each ACAT isoform was quantified by PCR from total sample cDNA using specific primers and TaqManTM probes normalised to beta-actin assayed in the same mutiplex reaction tube. This enabled us to calculate the relative abundance of the transcripts as an ACAT-1/ACAT-2 ratio. In liver (n=10), ACAT-1 transcripts were 43 times (range 7-125) more abundant than ACAT-2, whereas in duodenal samples (n=7) ACAT-2 transcripts were six- fold (range 2-11) greater than ACAT-1. Expressed as a percentage of total ACAT mRNA, human liver therefore comprises 98% ACAT-1 (range 86- 99%) whereas, the converse is true for human duodenum with 83% ACAT-2 (range 50-91%). These results demonstrate quantitatively for the first time that ACAT-1 is the predominate transcript in human liver, and ACAT-2 the most abundant in human duodenum.
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