Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 68)

Section 6 Data Analysis Module
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Where:
•
O
i
is the filtered value for a given data point, i
•
R
i
is the unfiltered value for data point i
• c is a weighted factor with a value of 2
• M is the arithmetic mean of all data points for the well within the given cycle.
The rolling boxcar filter is the arithmetic mean of the data readings i – (w–1), where w is the
filter width. For example, if you want to calculate the 20th data point (i=20) and the width is 4
(w=4), then you take the mean of the data points 17–20, data point 21 is the mean of the data
points 18–21, etc. The rolling boxcar filters only apply within a cycle. A global filter that smoothes
data from cycle to cycle is also available by clicking Enable Global Filter. The global filter operates
on the trace for a given well and fluorophore using all cycles together in a single pass. Global
filtering should be reserved for data that appears very noisy and should not be applied routinely.
Base Line Threshold Settings
In the amplification chart, the iQ5 software uses the baselines for each individual trace and the
threshold that is set for the fluorophore to set conditions to determine the threshold cycle.
Baseline cycles and threshold are only calculated by the software when in PCR Base Line
Subtracted or PCR Baseline Subtracted Curve Fit mode. You can override the automatic
conditions set for baseline and threshold in the Base Line Threshold window.
1. Ensure that only one fluorophore from the Amp Chart Fluorophore Selector buttons is
selected
2. Right-click on the amplification chart
3. Click Base Line Threshold. After you click Base Line Threshold in the amplification plot
menu, the Base Line Threshold Parameter window appears and displays the fluorophore
of the traces to be adjusted.
4. Select User Defined. In Figure 6.15, the Base Line Threshold Parameter window shows
the FAM traces are to be adjusted.
Fig. 6.15. The Base Line Threshold Parameter Window.