Chapter 9 Analyze the Comparative C
T
Experiment
View the Analysis Settings
177
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
Analysis
Guidelines
Unless you have already determined the optimal settings for your experiment, use the
default analysis settings in the 7500 software. If the default settings are not suitable for
your experiment, you can change the:
• C
T
Settings – Use this tab to manually set the threshold and baseline. When
manually setting the threshold and baseline, Applied Biosystems recommends the
following:
• Flag Settings – Use this tab to:
– Adjust the sensitivity so that more wells or fewer wells are flagged.
– Change the flags that are applied by the 7500 software.
• Relative Quantitation Settings – Use this tab to:
– Select the type of analysis to perform.
– Select the reference sample(s).
– Select the endogenous control. You can set more than one endogenous control.
See “Set Multiple Endogenous Controls” below.
– Set the efficiency. See “Set Efficiency” on page 178.
– Reject outliers.
– Select the algorithm to use to determine RQ Min/Max values (confidence level or
standard deviations).
• Advanced Settings – Use this tab to change baseline settings well by well.
Set Multiple
Endogenous
Controls
You can select multiple endogenous controls for a comparative C
T
experiment.
To select multiple controls:
1. In the Analysis Settings for Comparative Ct Example dialog box, select the Relative
Quantitation Settings tab.
2. In the Select Endogenous Control(s) group, click the Endogenous Control(s) drop-
down list, then select Multiple Controls.
Setting Recommendation
Threshold Enter a value for the threshold so that the threshold is:
• Above the background.
• Below the plateau and linear regions of the amplification curve.
• Within the exponential phase of the amplification curve.
Baseline Select the Start Cycle and End Cycle values so that the baseline
ends before significant fluorescent signal is detected.
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