II!I!I Applications in Oncology
Expression Analysis
of the Leukemia
Antigen PRAME
and of the HouseKeeping Genes
I3-Actin and TBP
by Real-Time PCR
• Melting Curve Analysis
Parameter
Value
Cycles
Type
Target temperature [0C]
Incubation time [s1
Temperature transition rate [OC/s]
Acquisition mode
Results
Melting Curve
Segment 1
9S
0
20
None
Segment 2
Segment 3
58
9S
10
0
20
0.1
None
ConI.
To quantify the mRNA expression of PRAME, we established the real-time RTPCR using the LightCycler SYBR Green I format. For the RT-PCR, MgCl2 concentrations of 1.625, 2.0, 2.25 and 3 mM were evaluated. Best results were obtained
with a concentration of 2.0 mM MgCI2, so this concentration was used in further
experiments. Figure la,b shows the sensitivity of the LightCycler RT-PCR detecting PRAME-positive leukemia cells in a sample of PRAME-negative mononuclear
peripheral blood cells of a healthy volunteer. Normal mononuclear cells of
peripheral blood showed no expression of PRAME. One PRAME-positive
leukemia cell of the leukemia cell line K562 in 1 X 10 5 normal cells was detectable.
Figure la shows the amplification and Fig. 1 b the melting curve data of K562 cells
diluted in normal mononuclear cells.
The quantitative expression of the antigen PRAME was evaluated using serial
dilutions of a PRAME RT-PCR product. Figure 2a shows the amplification curves
of different amounts of PRAME cDNA. The product was prepared by isolation
(Qiaquick PCR Purification Kit, Qiagen) of a conventional RT-PCR product of
PRAME. The concentration of the amplified DNA was measured by the photometer. The starting amounts of PRAME cDNA (11-0.11 pg) were used for preparation of standard curves. These curves were used as standard curves for the quantification of PRAME mRNA expression in cell lines and patients. The amplification of 50 ng of a K562 cDNA showed 2.5 pg of PRAME amplification starting
product (sample 6). The melting curves of the quantification standard curves and
of a K562 PCR product are shown in Fig. 2b. All melting curves showed a PRAMEspecific peak at 90°C.
The mRNA expression of PRAME was correlated with the mRNA expression of
the house-keeping genes ~-actin and TBP (no retro-pseudogenes known [9]). The
LightCycler RT-PCR of these genes was established. Figure 3 shows the amplification data of the house-keeping gene ~-actin. Different MgCl2 concentrations
(1.625; 2.25; 3.0 and 4.0 mM) were tested for ~-actin RT-PCR. For further experiments a MgCl 2 concentration of 2.25 mM was used. Figure 4a shows the amplification curves of serial diluted cDNA samples of the house-keeping gene TBP.
These samples were prepared by isolation of a conventional RT-PCR product of
Expression Analysis
of the Leukemia
Antigen PRAME
and of the HouseKeeping Genes
I3-Actin and TBP
by Real-Time PCR
• Melting Curve Analysis
Parameter
Value
Cycles
Type
Target temperature [0C]
Incubation time [s1
Temperature transition rate [OC/s]
Acquisition mode
Results
Melting Curve
Segment 1
9S
0
20
None
Segment 2
Segment 3
58
9S
10
0
20
0.1
None
ConI.
To quantify the mRNA expression of PRAME, we established the real-time RTPCR using the LightCycler SYBR Green I format. For the RT-PCR, MgCl2 concentrations of 1.625, 2.0, 2.25 and 3 mM were evaluated. Best results were obtained
with a concentration of 2.0 mM MgCI2, so this concentration was used in further
experiments. Figure la,b shows the sensitivity of the LightCycler RT-PCR detecting PRAME-positive leukemia cells in a sample of PRAME-negative mononuclear
peripheral blood cells of a healthy volunteer. Normal mononuclear cells of
peripheral blood showed no expression of PRAME. One PRAME-positive
leukemia cell of the leukemia cell line K562 in 1 X 10 5 normal cells was detectable.
Figure la shows the amplification and Fig. 1 b the melting curve data of K562 cells
diluted in normal mononuclear cells.
The quantitative expression of the antigen PRAME was evaluated using serial
dilutions of a PRAME RT-PCR product. Figure 2a shows the amplification curves
of different amounts of PRAME cDNA. The product was prepared by isolation
(Qiaquick PCR Purification Kit, Qiagen) of a conventional RT-PCR product of
PRAME. The concentration of the amplified DNA was measured by the photometer. The starting amounts of PRAME cDNA (11-0.11 pg) were used for preparation of standard curves. These curves were used as standard curves for the quantification of PRAME mRNA expression in cell lines and patients. The amplification of 50 ng of a K562 cDNA showed 2.5 pg of PRAME amplification starting
product (sample 6). The melting curves of the quantification standard curves and
of a K562 PCR product are shown in Fig. 2b. All melting curves showed a PRAMEspecific peak at 90°C.
The mRNA expression of PRAME was correlated with the mRNA expression of
the house-keeping genes ~-actin and TBP (no retro-pseudogenes known [9]). The
LightCycler RT-PCR of these genes was established. Figure 3 shows the amplification data of the house-keeping gene ~-actin. Different MgCl2 concentrations
(1.625; 2.25; 3.0 and 4.0 mM) were tested for ~-actin RT-PCR. For further experiments a MgCl 2 concentration of 2.25 mM was used. Figure 4a shows the amplification curves of serial diluted cDNA samples of the house-keeping gene TBP.
These samples were prepared by isolation of a conventional RT-PCR product of