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Figure 5.3
Cq Calling:
The software defaults to the Cy0 method for Cq calling as it is recommended. You
may choose to select cpD2 for data analysis as the characteristics of the data itself can affect
how Cq methods perform.
Parameters:
The parameters section is set to default at the values shown in Figure 5.3. You
may customize as needed per each experiment.
Min. Fluorescence
– The minimum fluorescence threshold for Cq calling. Cq values will
not be called if fluorescence is below this threshold
Min. Cycle
– The earliest cycle to use in Cq calling and baseline subtraction. Data for
earlier cycles will be ignored
Min. 1
st
Derivative
– The threshold which the first derivative of the curve must exceed for
a Cq to be called
Min. 2
nd
Derivative
– The threshold which the second derivative of the curve must exceed
for a Cq to be called
Baseline Cycles
: Baseline refers to the background signal level prior to any significant product
amplification. The baseline should be wide enough to eliminate any background found in early
cycles of amplification.
Auto
– Allows the software to automatically detect the best baseline for each well
Manual
– Allows for manual adjustment for baseline cycles as appropriate. The baseline
will be the median fluorescence of the cycles you select.
BASELINE SUBTRACTION
The software’s baseline subtraction aligns the baseline of each curve. Baseline subtraction is
performed to reduce well-to-well variation so that there are similar levels of fluorescence
during the initial cycling stages.
