Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 59)

Section 6 Data Analysis Module
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Use the fluorophore selector buttons to select and deselect which fluorophores to display. De-
selecting a fluorophore removes it from the display, but not from the analysis, selected
fluorophore buttons are displayed with a red border around their perimeter. De-selected
fluorophores are displayed without this red border. In Figure 6.3, the FAM fluorophore has been
selected, and HEX has been de-selected.
Fig. 6.3. Fluorophore Selector Buttons.
6.2.2 Select Analysis Mode
You can select from three options in the Analysis Mode drop-down list box (Figure 6.4).
Fig. 6.4. The Analysis Mode Drop-Down List.
The three analysis modes include:
•
Background Subtracted: The background subtracted data is the relative fluorescence
of each fluorophore after normalizing for exposure time, background factors, and well
factors. No further analysis is possible on background subtracted data; therefore, the End
Point, Allelic Disc, and Gene Expr tabs are unavailable
• PCR Base Line Subtracted: To determine threshold cycles, construct standard curves,
and determine the concentration of unknown samples, the data must be PCR baseline
subtracted. The iQ5 software determines each PCR baseline subtracted trace by fitting
the best straight line through the recorded fluorescence of each well during the baseline
cycles. The iQ5 software then subtracts the best fit data from the background subtracted
data at each cycle to generate the PCR baseline subtracted trace. By default, the
software automatically chooses the beginning and end baseline cycles. You can override
this default and manually give each trace a beginning and ending baseline cycle. User
specified settings for PCR baseline subtraction may also be specified in the User
Preferences module
•
PCR Base Line Subtracted Curve Fit: The iQ5 software fits the PCR baseline
subtracted data to a smoothed curve using a balanced flank, centroid-finding digital filter.
The curve fit process is performed in such a way that the C
T
is left invariant for all traces
6.2.3 Log View Button
You can click Log View, shown below, to change between a semi-logarithmic and linear display
of the amplification chart data.