Biomedical Engineering Reference
In-Depth Information
Table 3
Compositions of Polymerase Chain Reaction Mix
for Amplification of hPL 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
)
1.5
300 n
M
Reverse primer (10
µ
M
)
1.5
300 n
M
Probe (5
µ
M
)
1
100 n
M
r
Tth
DNA polymerase (2.5 U/
µ
L)
2
0.1 U/
µ
L
AmpErase UNG (1 U/
µ
L)
0.5
0.01 U/
µ
L
RNase-free water
15.5
-
Total volume 44
hPL
;
human placental lactogen
; UNG,
uracil-
N
-glycosylase.
Table 4
Cycling Profile for Amplification of hPL 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
45 Cycles
Annealing/extension
56°C
1 min
hPL
;
human placental lactogen
;
UNG,
uracil-
N
-glycosylase.
3.3.3. Data Analysis
Amplification data were analyzed and stored by the Sequence Detection Sys-
tem Software (v1.9; Applied Biosystems). The
GAPDH
mRNA concentrations
are expressed as picogram of total RNA per milliliter of plasma (pg/mL)
whereas the
hPL
mRNA concentrations are expressed as copies of
hPL
tran-
scripts per milliliter of plasma (copies/mL). The calculation is shown as follows:
