Bio-Rad Firmware & Software Updates - Manual (Page 123)

Gene Expression Analysis
110
• Relative expression level of one target sequence in one sample compared to the
same target under different sample treatments. For example, the relative amount of
one gene relative to itself under different temporal, geographical, or developmental
conditions
Plate Setup for Gene Expression Analysis
To perform gene expression analysis, the contents of the wells must include the following:
• Two or more targets. The two targets that represent different amplified genes or
sequences in your samples
• One or more reference targets. At least one target must be a reference target for
normalized expression. Assign all reference targets in the Experiment Settings window
(page 53) to analyze the data in Normalized Expression mode (
ΔΔC
q
). Runs that do not
contain a reference must be analyzed using Relative Expression mode (
ΔC
q
)
• Common samples. Your reactions must include common samples (minimum of two
required) to view your data plotted in the Gene Expression tab. These samples represent
different treatments or conditions for each of your target sequences. Assign a control
sample (optional) in the Experiment Settings window (page 53). If no contol is selected, a
control value will be calculated using the average of the values from all samples.
The requirements for Gene Expression setup in the Plate Editor depend on whether reaction
contents are singleplex PCR, with one fluorophore in the reactions, or multiplex PCR, with
more than one fluorophore in the reactions.
Figure 80 shows an example of the minimum contents of the wells for a singleplex gene
expression run.
Figure 80. Example of well contents in a singleplex gene expression run.
Figure 81 shows an example of the minimum contents of the wells for a multiplex gene
expression run.
Figure 81. Example of well contents in a multiplex gene expression run.
Guided Plate Setup
If the plate setup of a data file does not contain the information required for analysis and the
Gene Expression tab is selected, the space normally occupied by the bar chart will contain
instructions for entering this information. For normalized gene expression, the following steps
must be completed:
1. Define Target and Sample names using
• Plate Setup. This will open the Plate Editor window