Biomedical Engineering Reference
In-Depth Information
Fig. 1. Amplification plot of the real-time polymerase chain reaction data. (1) Phase
of observed background fluorescence. (2) Observable exponential phase. (2a) Mea-
surements are close to the detection limit. As a result of the high contribution of the
background fluorescence to the total fluorescence measured, replicate curves can
deviate (as seen in the black curves). (2b) Influence of background fluorescence mini-
mal. (3) Linear and plateau phases of the amplification with decreasing amplification
efficiencies.
Displayed are triplicate amplifications of a sample with trisomy 21. The grey curves
represent data from chromosome 21 (FAM dye), the black curves from chromosome
18 (VIC dye).
In order for this analysis to be accurate, it is important that the co-amplifica-
tion of two sequences occur in the same reaction vessel. This scenario will
guarantee that no well-to-well variation occurs between the two amplified tar-
gets
(
19
)
. In this regard, it is important to realize that the analysis cannot be
carried out in a monoplex manner. As with any small well-to-well deviations
resulting from minute pipetting errors, alterations in polymerase activity, tem-
perature, or illumination gradients, as well as unequal reagent depletion, will
lead to an inaccurate assessment of the target chromosome ploidy. Similarly, it
is not possible to determine the ploidy of a sample by referring to a standard
curve, as this analysis will be misled by the same factors affecting monoplex
analyses, a facet which is not alleviated by post-run data analysis
(
22-25
)
.
In order to balance small fluctuations and ensure that the amplification of
both target sequences has proceeded with similar efficiency, we have devised
the following analytical aid: instead of a solitary C
T
-value, three to four points
