Techne PrimeQ - Manual (Page 137)

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• A1: Start temperature +10% ramp
• A2: Start temperature +30% ramp
For example, if the temperature ramps for 30°C between 50°C and 80°C, 10% would equate to
3°C and A1 would be positioned at 50 + 3 (53°C); 30% equates to 9°C and so A2 would be
positioned at 50 + 9 (59°C) and so on. These cursors should be positioned as far apart as possible
without including any of the dissociation data.
B1–B2: These cursors determine the remaining fluorescence background after the DNA has
dissociated. They are placed in the flat fluorescence region that occurs after the melt. The default
values are:
• B1: Start temperature +90% ramp
• B2: Start temperature +95% ramp
The cursors are applied to the fluorescence vs. temperature plot once the data has been collected.
Within the Results Editor, the user can adjust the cursors manually to best suit the data. The
background rate of change over the user-defined region A is calculated automatically for each well
using linear regression. This generates a correction index for each well which is used to correct
the data until it falls below a pre-defined point. The correction for region B is calculated taking the
average of all readings in region B and subtracting this value from the data for each well.
3.15.2.2 Peak detection
Clicking Next from the background correction screen leads to the peak detection options.
Principle
The plot of dissociation peaks involves a calculation that takes the negative of the rate of change
in fluorescence as a function of temperature (-dF/dT). This approach uses a second order
polynomial equation to calculate a best-fit curve through the data point plus one either side. The
rate of change for each value can then be calculated by differentiating the equation.
The highest point of each peak (Tm) can then be calculated using the second order polynomial
equation of the three points around the peak in a similar manner to the calculation for first
derivative maximum for Cq calculation in quantification analysis. The best-fit equation is
differentiated and solved for dy/dx = 0 to obtain temperature (Tm) at the peak.
Methods
The Dissociation Wizard provides two options for peak detection:
• The software is instructed to automatically find a number of peaks mathematically (Auto
peak detect).
• The user manually places cursors onto the graph of peak data (Manual peak detect).
3.15.2.3 Auto peak detect
The user defines the settings that the software will use to score for peaks.
• Number of peaks to find: Up to a maximum of four.
• Threshold filter: The user sets a threshold value to limit the size of the peaks detected (see
below). The default is 5%.
• Order by peak height or Tm: A list of peaks and height are produced for each well and
then filtered to remove any peaks less than the noise level. The results can be ordered
according to either the peak height (largest first) or temperature (Tm). If there are replicates,
each well will be treated individually.