Il1II Methods Useful in Genetics and Oncology
(5 mM), and 1 fig total RNA, as recommended by the manufacturer [1st Strand
cDNA Synthesis Kit for RT-PCR (AMV), Roche Molecular Biochemicals]. Serial
dilutions of in vitro synthesized ARCAl RNA (100 pg, 10 pg, 1 pg, 0.1 pg) were
reverse transcribed and 1/10 of each reaction was used for generation of a standard curve (Fig. 2). Following cDNA synthesis for 60 min at 42°C and inactivation
of the enzyme at 95°C for 5 min, PCR reactions were performed in the LightCycler instrument.
LightCycier PCR The following Hybridization Probe Master Mix was used for amplification of the
ARCA1- and PRGD-specific fragments. The same reaction mix was used for
amplification of the ABCAI RNA standard.
Volume [f!l]
[Final]
Hot Start PCR Reaction Mix
1.5
Ix
MgCl2 (25 mM)
2.4
5.0mM
Primers (25 J-lM each)
0.3+0.3
0.5 J-lM
Probes (20 J-lM each)
0.22+0.22
0.3 f.lM
H20 (PCR-grade)
8.06
Total volume
13
We added 13 fil of master mix and 2 f.Ll of RT reaction mixture (usually corresponding to 100 ng of total RNA) to each glass capillary. All of the reverse transcribed standard dilutions were amplified simultaneously. Sealed capillaries were
centrifuged briefly with the adapters in a micro centrifuge and put in the LightCycler carousel.
The following PCR protocol was used for amplification and melting curve
analysis:
• Denaturation
Parameter
Value
.
Cycles
Type
Target temperature [0C]
Incubation time [min]
Temperature transition rate [OC/s]
Acquisition Mode
Regular
Segment 1
95
10
20
None
(5 mM), and 1 fig total RNA, as recommended by the manufacturer [1st Strand
cDNA Synthesis Kit for RT-PCR (AMV), Roche Molecular Biochemicals]. Serial
dilutions of in vitro synthesized ARCAl RNA (100 pg, 10 pg, 1 pg, 0.1 pg) were
reverse transcribed and 1/10 of each reaction was used for generation of a standard curve (Fig. 2). Following cDNA synthesis for 60 min at 42°C and inactivation
of the enzyme at 95°C for 5 min, PCR reactions were performed in the LightCycler instrument.
LightCycier PCR The following Hybridization Probe Master Mix was used for amplification of the
ARCA1- and PRGD-specific fragments. The same reaction mix was used for
amplification of the ABCAI RNA standard.
Volume [f!l]
[Final]
Hot Start PCR Reaction Mix
1.5
Ix
MgCl2 (25 mM)
2.4
5.0mM
Primers (25 J-lM each)
0.3+0.3
0.5 J-lM
Probes (20 J-lM each)
0.22+0.22
0.3 f.lM
H20 (PCR-grade)
8.06
Total volume
13
We added 13 fil of master mix and 2 f.Ll of RT reaction mixture (usually corresponding to 100 ng of total RNA) to each glass capillary. All of the reverse transcribed standard dilutions were amplified simultaneously. Sealed capillaries were
centrifuged briefly with the adapters in a micro centrifuge and put in the LightCycler carousel.
The following PCR protocol was used for amplification and melting curve
analysis:
• Denaturation
Parameter
Value
.
Cycles
Type
Target temperature [0C]
Incubation time [min]
Temperature transition rate [OC/s]
Acquisition Mode
Regular
Segment 1
95
10
20
None