Use of Real-Time Polymerase Chain Reaction for the Detection of Fetal Aneuploidies - Clinical Applications of PCR - page 88

Fig. 2. Change in threshold cycle ( ∆ C T ) in the amplification plot (magnification of

Fig. 1 ). The ∆ C T s of the four thresholds, indicated by double-headed arrows.

are chosen along the linear phase of the amplification plot ( Fig. 2 ). Those
samples in which a deviation is found to occur at these points are either
discarded or reanalyzed. If this provision has been made, then the fluorescent
signals from both amplifications will be detected simultaneously if the sample
is karyotypically normal, i.e., both chromosomes being interrogated have the
same copy number ( Fig. 3 ). In case of a trisomy, the chromosome present at
three copies per cell will be detected at a lower (earlier) C T -value than the other.
The measured differences in C T (the

∆ C T ) between the two target chromo-

somes can be converted into a ratio ( 20 ) :
target A/target B = 2 -(∆C T ) = Chromosome 18/Chromosome 21
In theory, the difference in threshold cycle number (

C T ) for a normal (and
for a triploid) karyotype will be 0 cycle; for a trisomic sample, it will be ± 0.58
cycles, because 2 -(0.58) = 1.5
To correct for slight differences in reaction efficiencies and detector dye
intensities, the

∆

C T method, which relies on the analysis of a reference or
calibrator sample, can be used ( 21 ) . Normally, the reference or calibrator
sample is a sample of known (preferably normal) karyotype. The

∆∆

∆ C T of the

reference sample is subtracted from the C T of the sample, and then the cor-

rected ratio of the target sequences in the sample can be calculated as follows:
∆∆

∆

C T calibrated = C T (calibrator)

(target A/target B) calibrated = 2 -(∆∆C T calibrated)

∆ C T (sample) - ∆