Biomedical Engineering Reference
In-Depth Information
3.
In our experience, C
T
-values from 25.0 to 29.0 (for a threshold of 0.2) will yield
good results. C
T
-values of higher than 29.0 are associated with quantities of DNA
that are too low to guarantee that preferential amplification of one of the multi-
plexed reactions does not take place (
see
Note 22
).
4.
For every replicate, calculate the
∆
C
T
for each of the four thresholds (
Table 3
and
Fig. 2
).
5.
For every replicate and for all three ploidies that can be distinguished, calculate
the
∆∆
C
T
for each of the four thresholds:
∆∆
C
T
calibrated =
∆
C
T
(sample) -
∆
C
T
(calibrator).
6.
Calculate for every replicate, with respect to a normal karyotype and the two types
of trisomies, the average of the four
∆∆
C
T
-values.
7.
The analysis of one replicate that gives the value closest to 0 for the averaged
∆∆
C
T
is indicative of the chromosomal status. This value should be smaller than
±0.25 (
see
Note 23
); that is, for example:
∆∆
C
T
calibrated (trisomy 18 sample) =
∆
C
T
-
∆
C
T
(calibrator trisomy 18) = 0 ± 0.25.
8.
If the results of all three replicates are indicative of the same chromosomal bal-
ance, the ploidy of the sample is that of the matched reference
C
T
calibrators;
otherwise, the result is not conclusive but rather is suggestive (
see
Note 24
).
∆
9.
Alternatively, it is possible to add the four calibrated
C
T
-values. In this case, a
normal karyotype will have a value close to 0, whereas a trisomy 21 sample will
be on the order of 2 and a trisomy 18 sample will have a value of approx -2.
∆∆
4. Notes
1.
In order for this assay to work, the main requirement for the real-time PCR
instrument is that the fluorescence data are normalized to the passive reference
dye, such as the ROX dye used by ABI. Any instrument that offers this feature
should be suited for the test. In a previous study, we used the ABI PRISM 7700
Sequence Detection System successfully.
2.
The optical covers have a high light-transmission capacity, a necessity for highly
precise measurements. Although optical caps should also work (according to the
manufacturers' technical support), our experience shows that the automatic data
collection adjustments of the SDS7000 are not sufficient for achieving the accu-
racy needed. For the laser-illuminated SDS7700, the covers and caps work fine.
However, it is advisable to always use the same covering for the reaction wells,
because it has an influence on the measured fluorescence.
3.
The use of AmpErase UNG is absolutely required in order to avoid amplification
of any misprimed, nonspecific products formed prior to specific amplification. If
this step is omitted, the balance of the two multiplexed reactions can be lost and
one of the sequences will be amplified with a greater efficiency, resulting in false
results. For the same reason, use of a
Taq
DNA polymerase activated by Hot Start
is required.
4.
To match the two assays, a variety of primer combinations for the chromosome
21 were tested. For the sake of expedience, we have listed only the most efficient
ones as well as the pertinent MGB probes (
Table 2
).
