m Applications in Oncology
The housekeeping gene for both kits and the PBGD master mix for each 20-~
reaction:
Volume [!ill
[Final]
hTERTfhTR reaction mix
2
Ix
Reverse transcriptase (RT)
0.1
PBGD detection mix
2
IX
H 2 0 (peR grade)
13.9
Total volume
18
Capillaries needed
2+n
A total of 18 ~ of the appropriate master mix and 2 ~ of the sample RNA (100 ng
or 100-200 ng for hTERT or hTR, respectively) are added to each glass capillary
placed in cooled adapters, using the following pattern:
Capillary
Reaction
hTERT/hTR master mix
PBGD master mix
1
Standard 1
1 reaction
2
Standard 2
1 reaction
3
Standard 3
1 reaction
4
Standard 4
1 reaction
5
Standard 5
1 reaction
6
H20 control
1 reaction
7
Positive control
I reaction
8
H20 control
1 reaction
9
Positive control
1 reaction
10- 32
Samples of interest (n)
n reactions
n reactions
Number of reactions
7+n
2+n
The sealed capillaries are briefly centrifuged (lOs at 700 g) and put into the LightCycler rotor.
The following RT-PCR protocol is used:
• Reverse transcription at 60°C for 10 min
• Denaturation at 95°C for 30 s
• Amplification
Parameter
Value
Cycles
Type
Target temperature 1°C]
Incubation time [sl
Temperature transition rate [OC/s]
Acquisition mode
Gains
45 for hTR; 40 for hTERT
Quantification
Segment 1
Segment 2
95
o
20
60
10
20
None
Single
Fl=1;F2=15;F3=30
Segment 3
72
10
20
None