Quantitative Two-Step RT-PCR for the Detection of Human ABCAI Transporter on LightCyder EJ
after each run and then converted to derivative melting curves (Fig. 3a,b, middle
panels). The melting points (or T m , melting temperatures) serve to identify and
discriminate between both analyzed products (65°C and 70°C for ABCAl and
PBGD, respectively).
The ABCAI mRNA expression levels of various tissues were compared to
macrophages. The results are presented either as an absolute amount of ABCAl
mRNA based on a calculation using the standard curve (pg/sample) or are standardized by the quantification of PBGD (Fig. 3a,b, respectively, bottom panel).
From all samples examined, the highest levels of ABCAI mRNA were found in
macrophages, liver, and stomach. The lowest amounts of ABCAI transcripts were
detected in the pancreas and heart. Using our assay we have demonstrated ABCAI
mRNA up-regulation during adipocyte (2.5-fold increase) and monocyte differentiation (I.5-fold increase). We have found it very useful in exploring the effect
of cholesterol loading and deloading on ABCAl expression in macrophages and
THP-l cells. In addition, we were able to detect ABCAI mRNA in keratinocytes,
HaCaT cells and in stem cells isolated from cord blood, which previously was
impossible with conventional RT-PCR. With our quantitative RT-PCR we investigated the effect of Sp 1 and Sp3 transcription factors on ABCAI mRNA expression
in HeLa cells transfected. We found a dose-dependent up-regulation of ABCAI
transcript by SpI.
Comments
Quantification of the absolute ABCAl gene transcript number is a very important
issue in many studies related to its expression and regulation by metabolic factors, nutritional status and new antiatherogenic drug candidates. In the majority
of publications investigating ABCAl expression, conventional methods for gene
expression analysis were used, mainly Northern blot, a time-consuming and relatively less sensitive method that is difficult to standardize. Here we demonstrate
that a LightCycler-based ABCAI mRNA assay allows the amplification, detection
and quantification of minute amounts of ABCAI mRNA in a variety of tissues and
cells. Our hybridization probe assay not only makes it possible to estimate the
absolute amounts of ABCAl transcripts, but also allows determination of the relFig. 3a,b. Amplification, quantification and melting peak analysis of ABCAI (a) and PBGD (b)
PCR products from human tissues. Of total RNA, 1 Jlg from each tissue was reverse transcribed
and 2 J.1l aliquots of RT reaction mixture were amplified in the LightCycler in the presence of
hybridization probes. Melting curves were converted to derivative melting curves by plotting the
first negative derivative of the fluorescence with respect to the temperature (-dF/dT) against the
temperature. Melting temperatures (T m) identify each of the products (ABCAI T m =65°C; PBGD
Tm =70°C). No run-to-run variation in Tm values was observed. The absolute amounts (pg/sampie) of ABCAI transcripts were calculated as described in Fig. 2. The ABCAI/PBGD ratios were
calculated by dividing absolute amounts of ABCAI and PBGD determined for each sample. Bars
represent mean values (±SD) of four independent measurements: a, absolute amount of ABCAI
transcript (bottom); b,ABCAIIPBGD ratio (bottom)
after each run and then converted to derivative melting curves (Fig. 3a,b, middle
panels). The melting points (or T m , melting temperatures) serve to identify and
discriminate between both analyzed products (65°C and 70°C for ABCAl and
PBGD, respectively).
The ABCAI mRNA expression levels of various tissues were compared to
macrophages. The results are presented either as an absolute amount of ABCAl
mRNA based on a calculation using the standard curve (pg/sample) or are standardized by the quantification of PBGD (Fig. 3a,b, respectively, bottom panel).
From all samples examined, the highest levels of ABCAI mRNA were found in
macrophages, liver, and stomach. The lowest amounts of ABCAI transcripts were
detected in the pancreas and heart. Using our assay we have demonstrated ABCAI
mRNA up-regulation during adipocyte (2.5-fold increase) and monocyte differentiation (I.5-fold increase). We have found it very useful in exploring the effect
of cholesterol loading and deloading on ABCAl expression in macrophages and
THP-l cells. In addition, we were able to detect ABCAI mRNA in keratinocytes,
HaCaT cells and in stem cells isolated from cord blood, which previously was
impossible with conventional RT-PCR. With our quantitative RT-PCR we investigated the effect of Sp 1 and Sp3 transcription factors on ABCAI mRNA expression
in HeLa cells transfected. We found a dose-dependent up-regulation of ABCAI
transcript by SpI.
Comments
Quantification of the absolute ABCAl gene transcript number is a very important
issue in many studies related to its expression and regulation by metabolic factors, nutritional status and new antiatherogenic drug candidates. In the majority
of publications investigating ABCAl expression, conventional methods for gene
expression analysis were used, mainly Northern blot, a time-consuming and relatively less sensitive method that is difficult to standardize. Here we demonstrate
that a LightCycler-based ABCAI mRNA assay allows the amplification, detection
and quantification of minute amounts of ABCAI mRNA in a variety of tissues and
cells. Our hybridization probe assay not only makes it possible to estimate the
absolute amounts of ABCAl transcripts, but also allows determination of the relFig. 3a,b. Amplification, quantification and melting peak analysis of ABCAI (a) and PBGD (b)
PCR products from human tissues. Of total RNA, 1 Jlg from each tissue was reverse transcribed
and 2 J.1l aliquots of RT reaction mixture were amplified in the LightCycler in the presence of
hybridization probes. Melting curves were converted to derivative melting curves by plotting the
first negative derivative of the fluorescence with respect to the temperature (-dF/dT) against the
temperature. Melting temperatures (T m) identify each of the products (ABCAI T m =65°C; PBGD
Tm =70°C). No run-to-run variation in Tm values was observed. The absolute amounts (pg/sampie) of ABCAI transcripts were calculated as described in Fig. 2. The ABCAI/PBGD ratios were
calculated by dividing absolute amounts of ABCAI and PBGD determined for each sample. Bars
represent mean values (±SD) of four independent measurements: a, absolute amount of ABCAI
transcript (bottom); b,ABCAIIPBGD ratio (bottom)