Biomedical Engineering Reference
In-Depth Information
Table 1
Compositions of Polymerase Chain Reaction Mix
for Amplification of GAPDH mRNA
Volume for
Component
one reaction (
µ
L)
Final concentration
5X TaqMan EZ Buffer
10
1X
Mn(OAc)
2
(25 m
M
)
6
3 m
M
dATP (10 m
M
)
1.5
300
µ
M
dCTP (10 m
M
)
1.5
300
µ
M
dGTP (10 m
M
)
1.5
300
µ
M
dUTP (20 m
M
)
1.5
600
µ
M
Forward primer (10
µ
M
)
1
200 n
M
Reverse primer (10
µ
M
)
1
200 n
M
Probe (5
µ
M
)
1
100 n
M
r
Tth
DNA polymerase (2.5 U/mL)
2
0.1 U/
µ
L
AmpErase UNG (1 U/
µ
L)
0.5
0.01 U/
µ
L
RNase-free water
19.5
-
Total volume
47
GAPDH
,
glyceraldehyde-3-phosphate dehydrogenase;
UNG
,
uracil-
N
-glycosylase.
Table 2
Cycling Profile for Amplification of GAPDH mRNA
Step
Temperature
Time
UNG treatment
50°C
2 min
Reverse transcription
60°C
30 min
Deactivation of UNG
95°C
5 min
Denaturation
94°C
20 s
40 Cycles
Annealing/extension
62°C
1 min
GAPDH
,
glyceraldehyde-3-phosphate dehydrogenase
; UNG, uracil-
N
-glycosylase.
2.
Set up the RT-PCR reaction mixture for
hPL
mRNA according to
Table 3
.
3.
L of sample RNA, synthetic DNA oligonucleotides (for calibration curve)
or RNase-free water (for negative blanks) into the reaction mixture.
Add 6
µ
4.
Perform the real-time
hPL
RT-PCR reactions in the ABI Prism 7700 Sequence
Detector with cycling conditions as shown in
Table 4
.
