Biomedical Engineering Reference
In-Depth Information
6.2.5 qPCR using SYBR green I dye detection
There are numerous non-probe- and probe-based chemistries, described in detail elsewhere
[36]. The most suitable detection chemistry depends on the application; in general, the use
of a double-stranded DNA binding dye, typically SYBR Green I, is the most cost-effective
chemistry for primer optimization experiments and for most routine investigations [75]
(see Protocol 6.6). An example of a typical qPCR result obtained using SYBR Green I
dye as the reporter system is shown in Figure 6.3. Although inadvisable, legacy assays can
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Cycle
(a)
Figure 6.3
Comparative quantification. This procedure provides an alternative to the more
usual dilution curve analysis for determining the fold change of a gene of interest relative to a
calibrator sample. (a) Three amplification plots derived from a 100-fold serial dilution series of
an RNA template used to optimize a new primer set. Note that there is no threshold line, which
wouldbeusedtodeterminethe
C
q
using the usual dilution curve method. (b) How the relative
concentration of a sample is derived from the amplification plots shown in (a). (1) The second
derivatives of the three amplification plots are calculated. These produce peaks corresponding
to the maximum rate of fluorescence increase in the reaction denoted by 1, 2 and 3. (2) The
'takeoff' points (labelled 4, 5 and 6) are determined for each curve. A takeoff point is defined
as the cycle at which the second derivative is at 20% of the maximum level, and indicates the
end of the noise and the transition into the exponential phase. (3) The average increase in
signal four cycles following the takeoff point (denoted by three bars labeled a, b and c) is used
to calculate a slope, which provides a measure of the amplification efficiency for each curve. A
100% efficient reaction should double the signal in the exponential phase. So, if the signal was
10 at cycle 15, then went to 11 at cycle 16, it should go to 13 fluorescence units at cycle 17.
(4) All of the amplification values for each sample are averaged to give a mean efficiency of
a group of cycling curves for each sample (three in this example). The more variation there is
between the estimated amplification values of each sample, the larger the confidence interval
will be. In this example, the average amplification is 1.68
±
0.02 for the neat template and
1.76
±
0.01 and1.76
±
0.02 for the two dilutions. (5) The same procedure is carried out for
the calibrator sample and a fold change can then be calculated according to the formula Fold
change
=
Efficiency
(Calibrator takeoff-Target sample takeoff)
. (See Plate 6.3.)
