Techne PrimeQ - Manual (Page 120)

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3.14.2.1 PRD
If a PRD was assigned in the dye usage box, the next screen displays an option for passive
reference dye correction (see section 4.4). Clicking Next leads through to the Baseline Correction
screen.
3.14.2.2 Baseline correction
See section 4.5. Choose the method as required and set the values as appropriate.
3.14.2.3 Cq calculation
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 (Cq) is the point at which an amplification curve
intercepts the crossing line and measuring fluorescence at the Cq for each well will allow the most
reliable well-to-well comparison (and when compared to standards, the quantification of
unknowns). The Cq calculation screen provides two options for Cq calculation:
• Fit points: (Default option): Involves setting a noise and crossing threshold. Useful for
quantifying low copy number targets.
• First derivative maximum: Calculates Cq values automatically based on the point at which
the rate of change is fastest. As this method uses the shape of the curve to determine Cq,
there is no user-input in the analysis of the data. This method can be useful for analysing
high copy number templates (over 1000 copies).
3.14.2.4 Fit points
This approach sets a crossing line at a specified distance above a noise threshold. Where the
amplification curve intercepts the line, a specified number of data points above the line are used in
a linear regression calculation to determine the Cq.
Procedure:
• Select fit points method
• Set noise threshold
• Set crossing line
• Set number of fit points
• Select whether to display fit points on the graph