Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 90)

Section 6 Data Analysis Module
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3. 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
• Change the gene assignments in these wells by typing your desired name into the
gene pull-down men, then click enter to apply the name to the selected wells
• Change the condition assignments in these wells by typing your desired name into
the condition pull-down menu, then click enter to apply the name to the selected
wells
NOTE: The Gene and Condition Names have a character entry limit of 15 characters.
Minimize the gene expression plate interface by clicking on the “–” button to return to
the standard view of the Gene Expr tab window.
4. Click the Settings tab, then select Gene List to set your desired reference gene(s).
5. Click Normalized expression (ddCt).
6. Click Recalculate to see your results.
7. Normalized expression results are graphed. The Data Table spreadsheet is accessed by
clicking Data Table. The Data Table spreadsheet lists the Condition and Gene name,
calculated expression values and C
T
values. Right-click to print or export this data to
Excel.
NOTE: Use the Settings tab, then click Gene List to enter a specific user-defined gene
reaction efficiency. Use the Settings tab, then click Condition List to select a particular
sample as a control sample.
6.9.2 Relative Quantity (dC
T,
∆C
T
)
Relative quantity is the target sequence concentration relative to other samples in the
experiment. This is sequence quantity without taking into account reference genes for
normalization. No calculations are made to account for loading differences or differences in the
number of cells represented in each of your samples. Relative quantity can be viewed as non-
normalized expression. This value is sometimes referred to as dC
T
or ∆C
T
because of the equation
used to calculate relative quantity.
By definition, relative quantity data is not normalized. Typically researchers that do not use
reference genes are confident in one of the two following considerations:
•
Each condition (sample) assayed represents the same amount of biological sample.
Typically they choose to load the same mass of RNA or cDNA in each well and feel that
mass of nucleic acid is an effective way of normalizing the resulting data. No modification
of relative quantity data is needed to obtain normalized data. The data are normalized by
experimental design
or;
•
Any variance in the amount of biological sample loaded will be normalized in post-PCR
analysis by some method. For example, a researcher might choose to simply divide the
relative quantity value by the normalizing factor indicated after each of the examples
listed below. Options may include but are not limited to:
• Mass of nucleic acid loaded for each sample. Rel Quant/ng RNA represented in each
sample