Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 22)

Section 2 Quick Guides
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7. Choose between the Threshold Cycle and RFU display modes in the Display Mode area to
display the allelic discrimination data.
• Threshold Cycle displays the distribution of samples on the scatter plot based on the
threshold cycle (C
T
). Samples that do not cross threshold will be assigned the C
T
value
of the last cycle run in the experiment.
• RFU displays the distribution of samples on the scatter plot based on the RFU
generated by each sample at the last PCR cycle number. Click the drop-down list next
to the Select Cycle box to generate the scatter plot based on an RFU from a different
cycle of the PCR experiment.
8. Click Automatic Call or Manual Call. Automatic Call is the default parameter.
In Automatic Call: threshold bars are positioned automatically in one of two ways:
• Based on distribution of the control wells, when at least three wells have been
assigned to Control 1 and three to Control 2
• At 90 percent of the C T
range or 10 percent of the RFU range on each axis if no
controls are named
Manual Call is the alternative analysis mode. Adjustments may be made either in the
scatter plot or in the data spreadsheet:
• Scatter plot: Make a selection in the Allele Call box. Then click and drag the cursor
over the corresponding sample(s) in the scatter plot. The iQ5 software reassigns the
samples to the allele call selected by the radio button, and updates the data
spreadsheet accordingly
• Data spreadsheet: Click in the Call cell of the spreadsheet. A menu appears that lets
you select an allele call for that sample. Once you select an allele call, the scatter plot
reflects the change
NOTE: You may click and drag threshold bars directly on the plot to adjust Automatic
Call assignment.
9. Click Reports in the menu to obtain customized reports for the allelic discrimination
data.
2.4.4 Gene Expression Quick Guide
The iQ5 software can present expression data normalized to one or more reference genes, or, for
data normalized before PCR, as a relative quantity.
Calculating Relative Quantity (dC
T
, ∆C
T
)
To calculate Relative Quantity (dC
T
):
In the PCR Quant screen (with a .opd data file open), assess Threshold and Baseline information
for the data file and make changes if necessary.
1. Click the Gene Expr tab.
2. Make any changes to Gene and Condition (for example, Sample and Treatment)
assignments in the Gene Expression Plate interface.
•
Expand the Gene Expression Plate Interface view by clicking on the “+” button to
make well identification and selection easier. Highlight the wells in the gene
expression plate interface you wish to edit