abi 7500 - Set up the Relative Quantitation Settings

Chapter 6 Design the Comparative C
T
Experiment
Set Up the Relative Quantitation Settings
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
124
Notes
Set Up the Relative Quantitation Settings
In the Relative Quantitation Settings screen, select the reference sample and the
endogenous control to perform relative quantitation.
About the
Example
Experiment
In the comparative C
T
example experiment:
• Brain is used as the reference sample.
• 18S is used as the endogenous control.
Complete the
Relative
Quantitation
Settings Screen
1. In the Which sample do you want to use as the reference sample? drop-down list,
select Brain.
2. In the Which target do you want to use as the endogenous control? drop-down list,
select 18S.
3. Click Next.
Design
Guidelines
When you design your own comparative C
T
experiment:
• Select a reference sample from your previously created samples (“Set Up the
Samples” on page 121). Amplification results from the samples are compared to the
amplification results from the reference sample to determine relative expression.
• Select an endogenous control from your previously created target assays (“Set Up
the Targets” on page 119). Amplification results from the endogenous control are
used to normalize the amplification results from the target for differences in the
amount of template added to each reaction.
For More
Information
For more information on:
• Completing the Relative Quantitation Settings screen – Open the 7500 Software
Help by clicking or pressing F1.
• Reference samples (also known as calibrators) and endogenous controls – See User
Bulletin #2: Relative Quantitation of Gene Expression.
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