Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 108)

Section 6 Data Analysis Module
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How does normalized expression as calculated by this software compare to the model
introduced by Dr. M. Pfaffl, et al.
If you only evaluate one reference gene and one gene of interest you will get exactly the same
results using the iQ5 software as you would using the model introduced in this paper. Standard
deviations may be slightly different.
How does normalized expression as calculated by this software compare to the model
outlined by Dr. Jo Vandesompele on the geNorm Web site?
The iQ5 software uses the models outline on the geNorm web site
(http://medgen.ugent.be/~jvdesomp/genorm/) and gives you the same results.
Why would I have to assign gene names in the Gene Expr tab?
If you have not assigned gene names in your initial plate setup or if you are studying more than
5 genes, you can use the plate interface in the Gene Expr tab to assign these relationships.
Can I customize my gene and condition names?
Yes. Both the gene and condition pull-down menus will accept any text you type into them. If
your names are very long, you can edit the name field in the gene and condition lists. The long
name will only be displayed in the legend above the graph. The name is displayed on the x-axis.
How do I determine reaction efficiencies?
Typically the efficiency for each primer (or primer/probe) set is evaluated and recorded during
assay development. Generate a standard curve using serial dilutions of a representative sample
across a relevant dynamic range, and then record the efficiency for subsequent gene expression
analysis.
6.10 Gene Study: Multi File Gene Expression Analysis
When conducting gene expression experiments, it may become necessary to run more than a
single plate in order to analyze all required samples. Similarly, the experimental goal may be to
analyze samples/conditions over a fixed time course. In both cases, it is essential to perform
gene expression analysis on data generated in different data sets. To accommodate this need, a
Gene Study file can be created in the iQ5 software.
6.10.1 The Gene Study File
A Gene Study file is a specialized file consisting of data and sample information imported from
multiple .opd files. The imported data is grouped into a single study, which can be edited (files
added or removed) and further analyzed at the users discretion. Gene Study files are assigned a
file extension of .gxd by the iQ5 software.
A Gene Study file is capable of comparing approximately 5,000 total wells of data. This implies a
maximum comparison of approximately 52 .opd files containing 96 wells of single-fluor (non-
multiplexed) data, or 10 .opd files containing 96 wells of five-fluor (multiplexed) data. Many other
combinations of file number, well capacity, and fluor number are possible. The absolute well
maximum will depend on the amount of RAM and virtual memory available to your computer.
Enable for Gene Study Button
All .opd files must be enabled for gene study analysis from within the Data Analysis module of
the iQ5 software. Enabling an .opd file for gene study analysis allows the iQ5 software to extract
only the information critical to gene expression analysis between different files. This step is