Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 89)

Section 6 Data Analysis Module
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Basic Workflow Steps for Gene Expression Analysis
1. 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.
2. Click the Gene Expr tab.
3. Make any changes to the Gene and Condition (for example, Sample and Treatment)
assignments in the gene expression plate interface.
4. Set reference genes if required, and choose an analysis method:
• Normalized Expression (ddC
T
) is the default; or
• Relative Quantity (dC
T
)
6. OPTIONAL: Assign attributes (sample color, show graph, etc.) and user-defined
reaction efficiencies in the Gene List.
7. OPTIONAL: Assign attributes (sample color, show graph, etc.) and set control samples
in the Condition List.
8. Click Recalculate to see your results.
If you want to compare these data to results obtained in other .opd files, you will need to enable
this file for Multi-file Gene Expression analysis, also called a Gene Study. To enable your file for
Gene Study:
1. Click Enable for Gene Study.
2. Go to the File menu to save your file.
Normalized expression is graphed in the gene expression graph interface. The data can be
accessed in the Gene Expression Settings interface within the Data Table.
6.9.1 Normalized Gene Expression (ddC
T,
∆∆C
T
)
Rather than using some other method to normalize data, you may use the measured expression
level of one or more reference genes as a normalization factor. Reference genes should be genes
which are not regulated in the biological system being studied.
Normalized expression is the relative quantity of your gene normalized to the relative quantities
of the reference gene(s). Simply stated, the quantities of the reference genes are used to
normalize the values of your genes of interest.
Provided the reference genes are not regulated in your system, normalized expression
calculations will account for loading differences or variations in cell number represented in each
of your samples. This value is sometimes referred to as ddC
T
or ∆∆C
T
because of the equation
initially introduced by Livak et al. (1995) to evaluate normalized expression. The software uses a
modification of this equation.
Normalized expression can be viewed as normalized target sequence quantity if your assay has
included controls for this purpose.
To calculate Normalized Expression (ddC
T
):
1. 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.
2. Click the Gene Expr tab.