Biomedical Engineering Reference
In-Depth Information
quence, the calculated chromosome ratios will average 1.4 rather than 1.5 for a
trisomic sample. Although the exact reason for this small anomaly is unclear, it
is likely attributable to minor differences in amplification efficiencies or
incomplete spectral separation of the dyes ( 26 ) .
Because the
C T -values themselves are indicative of the karyotype, i.e.,
close to 0 for normal and ±0.58 depending on the type of trisomy, it is not
actually necessary to determine the ratio. This is particularly evident when
taking an average
∆∆
C T -value taken at the four independent threshold points
( Table 1 ). Alternatively, it is possible to add these four independent
∆∆
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. In this manner, it is possible to determine the karyotypes
of the samples with a minimum of postrun work by setting the cut-off range
around the average
∆∆
C T of a given karyotype. In our analyses, we therefore
set a cut-off range of 0.25 cycle around the average measured
∆∆
∆∆
C T of a given
karyotype, because the measured
∆∆
C T s between normal and trisomy are
C T calibrated of karyotypically normal samples
will have a range of 0.00 ± 0.25. The accuracy of the test result can be
confirmed by reanalyzing the suspected trisomic sample in comparison with a
reference value made up of matched aneuploid samples. Furthermore, the test
can be made more stringent by defining a smaller range.
An example of such a
approx ±0.5. Accordingly, the
∆∆
C T analysis, where trisomy 18 and 21 samples
could be discriminated from one another as well as samples with normal ploidy
for these chromosomes, is illustrated as a scatter plot in Fig. 3 . This figure
shows the clear segregation of the three different karyotypes. In addition, the
manner in which the averaged
∆∆
C T -values of all relevant control samples can
serve as calibrator or reference values is displayed in Table 1 . In our hands,
this simple procedure (and its modifications as described in under Subheading
3. ) has been shown to be most reliable, producing consistent results over sev-
eral experiments and months.
1.3. Differences Between the Use of the SDS 7700
and SDS 7000 Real-Time PCR “Taqman” Instruments
In our proof-of-principle study for the detection of trisomy 21 by real-time
PCR, we used the Applied Biosystems Sequence Detection System 7700 (SDS
7700) instrument, which employs a complex laser array for the excitation of
the fluorescent-labeled reporter molecules. In this study, in which we exam-
ined only a small number of samples ( n = 21), we were able to correctly deter-
mine the karyotype in 10 of the 11 trisomy 21 samples analyzed. The analysis
also included 10 dilutions of the trisomy 21 samples. In two of the control
samples and in one sample with trisomy 21, no definitive assessment was pos-
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