Chapter 6 Design the Comparative C
T
Experiment
Set Up the Samples
121
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
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 comparative C
T
example experiment:
• Four samples are used: cDNA prepared from total RNA isolated from four tissues
(brain, kidney, liver, and universal). The samples contain unknown quantities of
EGR3, MAOB, OGDH, OSGEP, and SERPING1 (targets), and 18S (endogenous
control).
• Four technical replicates are used. The replicates are identical reactions, containing
identical reaction components and volumes.
• No negative controls are used. Negative control reactions contain water instead of
sample and should not amplify. The software automatically includes three negative
controls for each target assay.
IMPORTANT! Although the example experiment does not use negative controls,
Applied Biosystems recommends them.
Complete the
Samples Screen
1. Click the How many samples do you want to test in the reaction plate? field, then
enter 4.
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 4.
3. Click the How many negative controls do you need for each target assay? field,
then enter 0.
4. Set up Sample 1:
a. Click the Enter Sample Name field, then enter Brain.
b. Leave the default in the Color field.
5. Set up the remaining samples as shown below:
6. Select No Biological Replicates.
7. Select All Sample/Target Reactions to test all targets in all samples.
Sample Name Color
Kidney (Use default color)
Liver (Use default color)
Universal (Use default color)
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