J. Lipid Res.
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A more recent version of this article appeared on April 1, 2004

Papers In Press, published online ahead of print January 16, 2004
J. Lipid Res., doi:10.1194/jlr.M300365-JLR200
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Submitted on August 29, 2003
Revised on December 3, 2003
Accepted on January 5, 2004

Quantitative analysis of expression of ACAT genes in human tissues by real-time PCR

Jeffery L. Smith, Kavitha Rangaraj, Robert Simpson, Donald J. Maclean, Les K. Nathanson, Katherine A. Stuart, Shaun P. Scott, Grant A. Ramm, and John de Jersey

Biochemistry and Molecular Biology, The University of Queensland, Brisbane, Queensland 4072

Corresponding Author: jefferysmith{at}optusnet.com.au

Acyl-coenzyme A:cholesterol acyltransferase (ACAT, also called SOAT) catalyses the esterification of cholesterol by reaction with long chain acyl-coenzyme A derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after reverse transcriptase (RT) treatment of total RNA using TaqManTM real-time, quantitive PCR (RT-qPCR) normalised to beta -actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n=17), ACAT-1 transcripts were on average 9-fold (range 1.7 to 167) more abundant than ACAT-2, whereas in duodenal samples (n=10), ACAT-2 transcripts were on average 3-fold (range 0.39 to 12.2) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in sub-group analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver, and ACAT-2 transcripts predominate in human duodenum, and support the notion that ACAT-2 has an important regulatory role in liver and intestine.


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