Docstoc

667

Document Sample
667 Powered By Docstoc
					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.

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:2
posted:9/27/2011
language:English
pages:2