Rapid quantification of human ABCA1 mRNA in various cell types and tissues by real-time reverse transcription-PCR.
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 cholesterol and phospholipids; therefore, quantification of the ABCA1 mRNA is critical in many studies related to its expression and regulation by metabolic factors, nutritional status, and new antiatherogenic drug candidates. We developed a rapid, sensitive, specific, and reproducible real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of ABCA1 transcripts 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-transcribed 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 calculated from the calibration curve. In addition, normalized values of target (ABCA1/PBGD ratio) were calculated.


RESULTS
Using this method, we quantified ABCA1 transcripts in various human tissue samples as well as in monocytes, THP-1 cells, fibroblasts, and adipocytes. We demonstrated ABCA1 mRNA up-regulation during human adipocyte and monocyte differentiation. In addition, 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 Northern blot; it therefore could simplify all studies on ABCA1 mRNA expression.
Kielar
D
D
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, 93042 Regensburg, Germany.
Dietmaier
W
W
Langmann
T
T
Aslanidis
C
C
Probst
M
M
Naruszewicz
M
M
Schmitz
G
G
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D017667:Adipocytes / Q000737:chemistry; D015152:Blotting, Northern; D002478:Cells, Cultured; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D009000:Monocytes / Q000737:chemistry; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D012333:RNA, Messenger