Chapter 2 Design the Relative Standard Curve Experiment
Set Up the Samples
31
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
For More
Information
For more information on:
• Completing the Standards screen – Open the 7500 Software Help by clicking or
pressing F1.
• Amplification efficiency – Refer to the Amplification Efficiency of TaqMan
®
Gene
Expression Assays Application Note.
Set Up the Samples
In the Samples screen, enter the number of samples, replicates, and negative controls to
include in the reaction plate, enter the sample names, then select the sample/target
reactions to set up.
About the
Example
Experiment
In the relative standard curve example experiment:
• Two samples are used: cDNA prepared from total RNA isolated from liver and
kidney tissues. The samples contain unknown quantities of the c-myc gene (target)
and GAPDH gene (endogenous control).
• Three replicates are used. The replicates are identical reactions, containing identical
reaction components and volumes.
• Three negative controls are used. The negative control reactions contain water
instead of sample and should not amplify.
Complete the
Samples Screen
1. In the Samples screen, click the How many samples do you want to test in the
reaction plate? field, then enter 2.
Note: The number of rows in the samples table is updated with the number you
entered.
2. Click the How many replicates do you need? field, then enter 3.
3. Click the How many negative controls do you need for each target assay? field,
then enter 3.
4. Set up Sample 1:
a. Click the Enter Sample Name field, then enter Liver.
b. In the Color field, leave the default.
5. Set up Sample 2:
a. Click the Enter Sample Name field, then enter Kidney.
b. In the Color field, leave the default.
6. Select No Biological Replicates.
7. Select All Sample/Target Reactions to test all targets in all samples.
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