Biomedical Engineering Reference
In-Depth Information
the same. Otherwise, the optimization of the assays would be very difficult and
the sensitivities of the assays would have wide variations (
see
Note 3
). As the
melting temperatures of all the primers were very close, the thermal cycles for all
the assays were identical.
3.3.2. Establishing the Calibration Standards With Known Amounts
of EBV DNA
1.
For the quantification of circulating EBV DNA, a calibration curve must be estab-
lished. DNA solutions with known quantities of EBV DNA could be prepared
from the Namalwa cell line
(
13
)
(
see
Note 4
).
2.
Harvest Namalwa cells following tissue culture.
3.
Extract the DNA from these cells using the QIAamp Mini Kit following the blood
and body fluid protocol.
4.
Measure the concentration of total DNA (
D
µ
g/mL) by spectrophotometry.
5.
Translate the concentration of total DNA into EBV genome equivalents in each
microliter of solution (
N
genomes/5
µ
L) by the following formula (
see
Note 4
):
N
=
D
×
1000
×
2 Y 5/6.6
6.
Dilute the DNA solution to obtain a standard concentration of 100,000 genomes/
µ
L) serially to obtain DNA solu-
tions with concentrations of 10,000, 1000, 500, 100, 50, 25, 12.5, 6.25, 3.13, 1.57,
and 0.78 genomes/
L. Then, dilute the standard (100,000 genomes/
µ
µ
L.
3.3.3. Real-Time PCR for Assays With Differently Sized Amplicons
1.
Prepare the PCR master mix according to the protocol in stated
Table 2
(
see
Notes 5
and
6
).
2.
Pipet 45
µ
L of master mix into each well of a MicroAmp Optical 96-well reac-
tion plate.
3.
Pipet 5
L of each sample (including the calibration standards (
see
Notes 7
and
8
)
and plasma DNA samples of unknown concentrations) into each well in which the
master mix has been added in duplicate.
µ
4.
Seal the 96-well reaction plate with optical caps.
5.
Put the 96-well reaction plate in the reaction chamber of ABI 7700 sequence
detector.
6.
Input the sample names and types to the sequence detector program and start the
program.
7.
The real-time PCR reactions are carried out in an Applied Biosystem 7700
Sequence Detector with a thermal profile as stated in
Table 3
.
3.4. Presentation of Results
Real-time PCR assays with amplicons sized 82 to 382 bp were able to detect
1 copy of EBV DNA per reaction, whereas those with amplicons sized 493 to
1000 bp were able to detect 10 copies of EBV DNA per reaction. The plot of
plasma EBV DNA concentrations in NPC patients is shown in
Fig. 2B
. As the
