Rapid Quantification of Human ABCA1 mRNA in Various Cell Types and Tissues by Real-Time Reverse Transcription-PCR Danuta Kielar, 1,3 Wolfgang Dietmaier, 2 Thomas Langmann, 1 Charalampos Aslanidis, 1 Mario Probst, 1 Marek Naruszewicz, 3 and Gerd Schmitz 1* Background: The ABCA1 gene encodes for a member of subfamily A of the ATP-binding cassette transporters that plays an important role in cellular export of choles- terol and phospholipids; therefore, quantification of the ABCA1 mRNA is critical in many studies related to its expression and regulation by metabolic factors, nutri- tional status, and new antiatherogenic drug candidates. We developed a rapid, sensitive, specific, and reproduc- ible real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of ABCA1 tran- scripts in total RNA isolated from cultured human cells and tissues. Methods: To quantify ABCA1 mRNA, we generated a calibration curve from serial dilutions of in vitro-tran- scribed RNA corresponding to an amplified ABCA1 cDNA 205-bp fragment (homologous calibrator). Two pairs of fluorescent hybridization probes were used to detect the ABCA1 and porphobilinogen deaminase (PBGD) mRNAs; the latter served as an internal control. PCR was performed as real-time amplification of ABCA1 mRNA in 100 ng of total RNA isolated from various human tissues, and cultured cells were calcu- lated from the calibration curve. In addition, normalized values of target (ABCA1/PBGD ratio) were calculated. Results: Using this method, we quantified ABCA1 tran- scripts in various human tissue samples as well as in monocytes, THP-1 cells, fibroblasts, and adipocytes. We demonstrated ABCA1 mRNA up-regulation during hu- man adipocyte and monocyte differentiation. In addi- tion, we examined the effect of cholesterol loading and deloading on ABCA1 expression in monocytes, THP-1 cells, and fibroblasts. Conclusions: Our RT-PCR assay allows the specific and highly reproducible detection and quantification of minute amounts of human ABCA1 mRNA. This new method is more accurate, more informative, and less laborious than the classic RT-PCR methods and North- ern blot; it therefore could simplify all studies on ABCA1 mRNA expression. © 2001 American Association for Clinical Chemistry Cholesterol homeostasis in biologic systems and individual cells is maintained by mechanisms regulating its synthesis, transport, and degradation. The latter process takes place in the liver where it is metabolized to bile acids and subse- quently excreted. From peripheral tissues, cholesterol is transported by HDL particles, which protect cells, especially arterial wall macrophages, against sterol overload, a well- known causative factor in atherogenesis. ATP-binding cas- sette transporter A1 (ABCA1) 4 is a member of subfamily A of the ATP-binding cassette transporters, which cause famil- ial HDL deficiency syndromes (1–3 ). Initial evidence that ABCA1 is involved in cholesterol and phospholipid trans- port came from experiments in macrophages demonstrating that ABCA1 expression is sensitive to intracellular choles- terol concentrations (4 ). ABCA1 mRNA and protein in human macrophages are up-regulated by cholesterol load- ing and down-regulated by cholesterol deloading. The bio- logic importance of ABCA1 was further substantiated by observations that the absence of ABCA1 protein in patients 1 Institute for Clinical Chemistry and Laboratory Medicine and 2 Institute of Pathology, University of Regensburg, 93042 Regensburg, Germany. 3 Department of Clinical Biochemistry, Pomeranian Medical Academy, 70-111 Szczecin, Poland. *Address correspondence to this author at: Institute for Clinical Chemistry, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg, Germany. Fax 49-941-944-6202; e-mail gerd.schmitz@klinik.uni-regensburg.de. Received in revised form September 17, 2001; accepted September 18, 2001. 4 Nonstandard abbreviations: ABCA1, ATP-binding cassette transporter A1; RT-PCR, reverse transcription-PCR; M-CSF, macrophage colony-stimulat- ing factor; E-LDL, enzymatically modified LDL; PBGD, porphobilinogen deaminase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; and Cp, crossing point. Clinical Chemistry 47:12 2089 –2097 (2001) Molecular Diagnostics and Genetics 2089