Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 112)

Section 6 Data Analysis Module
- 103 -
corresponding row. If one or more files have been selected, clicking the Remove button will
remove those file(s) from the Gene Study.
The Notes field is also editable from the Gene Expression Study Manager. Once the desired
changes have been made, click OK to close the Gene Expression Study Manager and return to
analysis of the Gene Study file in the gene expression module. Remember to immediately save
your edits to the Gene Study before continuing with your analysis.
NOTE: The Edit Plate module of the iQ5 software is not available when a Gene Study file
(extension, .gxd) is opened. All plate information changes to the data in a Gene Study (that is.
sample ID), must be made from the plate interface of the Gene Expression window.
6.10.5 Excluding/Including Samples Using the Gene Expression Plate
Interface
When a Gene Study file is displayed, the Analyze Wells button that is available when viewing a
single Gene Expression file, is no longer displayed. In its place is the Include Sample/Exclude
Sample drop down list located above the plate interface (Figure 6.60).
Fig. 6.60. The Include Sample/Exclude Sample Selection Pull-Down Menu.
This drop down list works similar to Analyze Wells button (see Analyze Wells for details). To
exclude wells from the Gene Study analysis, select the specific wells from the plate interface.
Click on the drop down arrow box to select Exclude Sample from the list. The highlighted wells
will be grayed out to indicate that they are no longer included in the Gene Study analysis. To
include wells for the Gene Study analysis, select the specific wells from the plate interface. Click
on the drop down arrow box to select Include Sample from the list. The selected wells will be
displayed with the correct Gene and Condition Name colors to indicate that they are included in
the Gene Study analysis.
6.10.6 Inter-Run Calibration
Inter-run Calibration is unique to the Gene Study module. Inter-run Calibration is automatically
performed by the iQ5 software in every Gene Study. The resulting Inter-run Calibration
calculations are then used to normalize inter-run variations between genes assayed in separate
real-time PCR runs (that is, different .opd files).
The following conditions must be met for Inter-run Calibration:
•
A given sample must have the exact same Condition Name on each plate (per assay) and
for each gene – this allows the sample to be designated as an “inter-run calibrator”.
•
At least one “inter-run calibrator” sample must be present in the Gene Study.
When the above conditions are met, an algorithm is used to calculate the pair-wise differences
between the C
T
values (dC
T
) for all samples that qualify as inter-run calibrators (that is, having
the same Condition Name per gene and per assay).
Inter-run Calibration Algorithm
All data within the Gene Study is normalized to the inter-run calibrator that yields the smallest
average dC
T
value. The inter-run calibrator with the smallest average dC
T
value becomes the