Quantitative Two-Step RT-PCR for the Detection of Human
ABCA 1 Transporter on LightCycier Using Hybridization
Probes and External Standards
DANUTA KIELAR, WOLFGANG DIETMAIER, THOMAS LANG MANN,
CHARALAMPOS ASLANIDIS, MARIO PROBST, MAREK NARUSZEWICZ,
GERD SCHMITZ*
Introduction
ABCAI is a member of the subfamily A of ATP-binding cassette transporters,
whose recently discovered mutations cause familial HDL deficiency syndromes
such as Tangier disease [1-3]. Tangier patients have very low HDL levels, associated either with the classical phenotype (splenomegaly) or with premature atherosclerosis and a higher risk of developing coronary artery disease. Overexpression studies and analysis of ABCAI knock-out mice have demonstrated that
ABCA1 plays a key role in cellular transport of cholesterol and phospholipids [4,
5]. ABCA1 mRNA and protein in human macrophages are up-regulated by cholesterolloading and down-regulated by cholesterol deloading. Mechanisms controlling ABCAI gene expression comprise sterol-dependent and tissue-specific
pathways. ABCAI gene expression is up-regulated by modified LDL [6] cyclic
AMP [7,8] and oxysterols, which act on LXRa and LXR~ receptors together
with the retinoid X receptor (RXR) [9, 10]. A zinc finger transcription factor
(ZNF202) located within a hypoalphalipoproteinemia locus on chromosome
llq23 has a strong repressive capacity on ABCAI expression as well as cellular
lipid efflux [11]. However, the influence of other metabolic factors on the expression of ABCAI in humans is still not well understood. Therefore, a rapid, sensitive, specific and reproducible method for detection and quantification of
ABCA1 transcripts is needed. In this chapter, we describe a real-time RT-PCR
technique for detection and quantification of minute amounts of ABCA1 mRNA.
Using this method we quantified ABCA1 transcripts in various human tissues,
as well as in monocytes, macrophages, THP-1 cells, fibroblasts, adipocytes,
preadipocytes, keratinocytes and in HeLa cells transfected with Sp 1 and Sp3
transcription factors.
* Gerd Schmitz (~) (e-mail: gerd.schmitz@klinik.uni-regensburg.de)
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg,
Franz-J osef-Strauss-Allee 11, 93042 Regensburg, Germany
Wolfgang Dietmaier
Institute of Pathology University of Regensburg, 93042 Regensburg, Germany
Danuta Kielar, Marek Naruszewicz
Department of Clinical Biochemistry, Pomeranian Medical Academy, 70-111 Szczecin, Poland
ABCA 1 Transporter on LightCycier Using Hybridization
Probes and External Standards
DANUTA KIELAR, WOLFGANG DIETMAIER, THOMAS LANG MANN,
CHARALAMPOS ASLANIDIS, MARIO PROBST, MAREK NARUSZEWICZ,
GERD SCHMITZ*
Introduction
ABCAI is a member of the subfamily A of ATP-binding cassette transporters,
whose recently discovered mutations cause familial HDL deficiency syndromes
such as Tangier disease [1-3]. Tangier patients have very low HDL levels, associated either with the classical phenotype (splenomegaly) or with premature atherosclerosis and a higher risk of developing coronary artery disease. Overexpression studies and analysis of ABCAI knock-out mice have demonstrated that
ABCA1 plays a key role in cellular transport of cholesterol and phospholipids [4,
5]. ABCA1 mRNA and protein in human macrophages are up-regulated by cholesterolloading and down-regulated by cholesterol deloading. Mechanisms controlling ABCAI gene expression comprise sterol-dependent and tissue-specific
pathways. ABCAI gene expression is up-regulated by modified LDL [6] cyclic
AMP [7,8] and oxysterols, which act on LXRa and LXR~ receptors together
with the retinoid X receptor (RXR) [9, 10]. A zinc finger transcription factor
(ZNF202) located within a hypoalphalipoproteinemia locus on chromosome
llq23 has a strong repressive capacity on ABCAI expression as well as cellular
lipid efflux [11]. However, the influence of other metabolic factors on the expression of ABCAI in humans is still not well understood. Therefore, a rapid, sensitive, specific and reproducible method for detection and quantification of
ABCA1 transcripts is needed. In this chapter, we describe a real-time RT-PCR
technique for detection and quantification of minute amounts of ABCA1 mRNA.
Using this method we quantified ABCA1 transcripts in various human tissues,
as well as in monocytes, macrophages, THP-1 cells, fibroblasts, adipocytes,
preadipocytes, keratinocytes and in HeLa cells transfected with Sp 1 and Sp3
transcription factors.
* Gerd Schmitz (~) (e-mail: gerd.schmitz@klinik.uni-regensburg.de)
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg,
Franz-J osef-Strauss-Allee 11, 93042 Regensburg, Germany
Wolfgang Dietmaier
Institute of Pathology University of Regensburg, 93042 Regensburg, Germany
Danuta Kielar, Marek Naruszewicz
Department of Clinical Biochemistry, Pomeranian Medical Academy, 70-111 Szczecin, Poland