3 fit points – Techne PrimeQ User Manual
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The crossing line is a best-fit of where the reaction efficiency is at its highest and most constant
for each reaction curve. The quantification cycle, or Cq, (sometimes referred to as the C
T
in the
literature), is the point at which an amplification curve intercepts the crossing line. Measuring
fluorescence at the Cq for each well will allow the most reliable well-to-well comparison.
Plotting the Cq against the known concentration of standards will generate a standard curve that
makes it possible to estimate the starting concentration of unknowns.
The quantification cycle (Cq) is the point
where the amplification curve intercepts the
crossing line.
Two approaches are available for calculating the quantification cycle on PrimeQ: fit points and first
derivative maximum.
3.9.3 Fit points
This approach is the preferred approach when using low copy number templates and is based on
the setting of a crossing line and noise threshold. It is the default method used by Quansoft. The
noise threshold is used to define all non-informative fluorescence data – data that is typically
indistinguishable from the fluorescent noise seen in the early stages of amplification.
The crossing line is set at a specified number of standard deviations (SD) above the noise line
using either the default settings or those specified by the user. The default setting is 10 standard
deviations above the noise, which is the average of fluorescence measured in cycles 3–10.
Fluorescence is then plotted on a log scale vs. cycle number with the two cursors placed in the
appropriate positions: a blue line showing the noise threshold and a red, the crossing line.
The quantification cycle is then calculated using fit points. These are a defined number of
fluorescence points situated on the curve above the crossing line (default number is 2). The
software uses these points in a linear regression calculation to determine the best-fit gradient for
each well. The intersection of this line through the crossing line is the Cq and this will be a
fractional cycle number. The Cq is calculated for each well.
Note on log scale: The only real difference between a log and linear scale is the way the data is
displayed. The lower values in the log scale are ‘blown up’ to make the data more readable such
that the normalized values approaching zero can be seen more clearly. The small increase seen in
Cycle Number
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Cp
Cycle Number
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Cycle Number
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Cp
Cycle Number
Cycle Number
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High copy
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Low copy
numbers
Cp
Cycle Number
45
40
35
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25
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15
10
5
R
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High copy
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Low copy
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Cq