mJ Applications in Oncology
01 igonucleotide
Design
LightCycier PCR
The cDNA is synthesized from the telomerase positive reference sample and
diluted as follows: 1:1,1:2,1:4,1:8,1:16,1:32, and 1:64. Aliquots of 10 III are stored
at -20°C.
A quantity of 2 fll of each dilution is needed for each LightCycler run as quantity standards. Therefore, to have sufficient material for experiments, 4 fll of cDNA
should be produced for each expected LightCycler run.
Pairs of primers are designed for hTERT, hTR and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), used as a housekeeping gene (Table 1).
GAPDH master mix for each 20-fll reaction:
LightCycier-DNA Master SYBR Green I
MgCl 2 (25 roM)
Primers (10 IJM)
H20 (PCR grade)
Total volume
hTERT Master Mix for each 20-fll reaction:
LightCycler-DNA Master SYBR Green I
MgCl2 (25 mM)
Primers (10 IJM)
TaqStart Antibody
H20 (PCR grade)
Total volume
hTR master mix for each 20-fll reaction:
LightCyeler-DNA Master SYBR Green I
MgCl2 (25 roM)
Primers (10 ).1M)
TaqStart antibody
H20 (PCR grade)
Total volume
Volume If1Il
[Final)
2
1.6
1+1
12.4
18
Ix
3mM
0.51JM
Volume IIlI
[Final]
2
0.8
1+1
0.16
13.04
18
Ix
2mM
0.51JM
Volume If1I l
[Final)
2
I x
1.6
3mM
1+1
0.51JM
0.16
12.24
18
The relative GAPDH expression is determined prior to analyzing hTERT and hTR
expression.
The procedure for semiquantitative PCR on the LightCycler is similar for each
gene. A total of 18 fll of the respective master mix and 2 fll cDNA is added to each