Biomedical Engineering Reference
In-Depth Information
3.5. Real-Time Quantitative PCR Analysis (see Note 6)
1.
Prepare reagent master mix according to manufacturer's instructions for the
TaqMan PCR Core reagent kit (Applied Biosystems). The reagent mix consists
of 5
L 10X buffer A; 300 n
M
of each amplification primer, 100 n
M
of the corre-
sponding fluorescent probe, 4 m
M
MgCl
2
, 200
µ
µ
M
each dATP, dCTP, dGTP;
400
M
dUTP; 1.25 U Ampli
Taq
Gold; and 0.5 U AmpErase uracil
N
-glyco-
sylase
(2)
. Add dH
2
O to make up to a volume of 45
µ
µ
L per reaction. Thoroughly
mix the solution by vortexing.
2.
Dispense 45
L of reagent mix to each reaction well on a 96-well optical plate.
Perform duplicate analysis for each sample or standard (
see
Note 7
). Allow at
least two to four reactions as no-template control (NTC). Add known male and
female DNA samples as positive and negative controls.
µ
3.
Add 5
L of sample, standard or dH
2
O (for NTC), to the corresponding reac-
tion well.
µ
4.
Take care to remove air bubbles from the sample-reagent mix before sealing the
optical plate. Ensure that the optical plate is sealed securely.
5.
Real-time quantitative PCR is carried out in an Applied Biosystems 7700
Sequence Detector. Set the thermal profile as follows: 2 min incubation at 50°C,
followed by a first denaturation of 10 min at 95°C, then 40 cycles of 95°C for
15 s and 60°C for 1 min
(
2
)
.
3.6. Data Interpretation
1.
Confirm that the NTC and negative controls are negative. Confirm that the posi-
tive controls are positive. Assess the linearity of the standard curve by confirm-
ing whether the correlation coefficient is better than 0.95. Confirm that the run
has a detection sensitivity of one to three copies. Proceed to further interpretation
of the PCR analysis when the above conditions are fulfilled.
2.
The number of copies of DNA template (
Q
) at the start of the reaction will be
computed by the sequence detection software (Applied Biosystems). Determine
the fetal (
SRY
) or maternal (
-globin
) DNA concentrations (
C
) in the original
maternal plasma samples by the following calculation:
β
V
1
DNA
CQ
=
×
×
(2)
V
V
ext
PCR
where
C
= DNA concentration in plasma (GE/mL);
Q
= plasma DNA quantity
(copies) determined by the sequence detector in a PCR;
V
DNA
= total volume of
plasma DNA obtained after extraction, typically 50
L per extraction;
V
PCR
=
volume of plasma DNA solution used for PCR, typically 5
µ
µ
L; and
V
ext
= vol-
ume of plasma extracted, typically 800
µ
L.
