Biomedical Engineering Reference
In-Depth Information
Fig. 3. Scatter plot of control DNAs for a threshold of 0.45. Depicted are replicate
averages per sample. The change in threshold cycle
(
C
T
)-values of all samples with
normal karyotype group between -0.22 and +0.10, all trisomy samples lie outside of
this area (trisomy 21 are above, trisomy 18 are below). The average
∆
∆
C
T
-value of the
C
T
(calibrator) for samples with a
normal karyotype at the threshold of 0.45 (
Table 1
).
normal samples is -0.09. This value is used as the
∆
C
T
-
value(s) of the analyzed sample, the use of a single reference sample can nev-
ertheless introduce a small source of error. This may be caused by minute
deviations in the amplification and signal generation, which may in turn lead to
the misinterpretation of the sample analyzed. In this regard, it is worth noting
that a seemingly small shift in the reference
Even though this method is more reliable than solely relying on the
∆
C
T
-value of only +0.15 cycle
from its “real” value may result in misdiagnosis of a sample with a similarly
small deviation of -0.15 cycle. For this reason, we recommend that for the
reliable determination of ploidy an adaptation of the
∆
C
T
method be used,
whereby the samples being analyzed are quantified relative to a reference
∆∆
∆
C
T
-
value determined from the average
C
T
-values of all karyotypically normal
samples tested previously. Furthermore, if this analysis suggests that a particu-
lar sample is aneuploid, then an added precaution may be to analyze this sample
using a reference comprised of similar aneuploid samples. In this manner, an
opposite pattern to that normally expected will emerge, in that the
∆
C
T
analy-
sis using a normal ploidy reference should be close to the theoretical value of
±0.58 cycle, whereas the
∆∆
C
T
comparison using a matched aneuploid refer-
ence standard should be close to 0.
In reality, however, the experimentally measured differences in
∆∆
C
T
between normal and trisomic karyotypes is not 0.58, but ranges from 0.47 to
0.55 depending on threshold-value set and the type of trisomy. As a conse-
∆∆
