Chapter 6
Design the Comparative C
T
Experiment
Set Up the Targets
119
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 Targets
In the Targets screen, enter the number of targets you want to quantify in the PCR
reaction plate, then set up the assay for each target.
About the
Example
Experiment
In the comparative C
T
example experiment:
• Two
targets are quantified in the reaction plate.
• The Target 1 assay is set up for the endogenous control. For the example experiment
the target is human 18S. 18S serves as the endogenous control because its expression
levels tend to be relatively stable.
• The Target 2 through 6 assays are set up for the targets you are studying. For the
example experiment, the targets are EGR3, MAOB, OGDH, OSGEP, and SERPING1.
Complete the
Targets Screen
1.
Click the
How many targets do you want to quantify in the reaction plate?
field,
then enter
6
.
Note:
The number of rows in the target assays table is updated with the number you
entered.
2.
Set up the Target 1 assay:
a.
Click the
Enter Target Name
cell, then enter
18S
.
b.
In the Reporter drop-down list, select
FAM
(default).
c.
In the Quencher drop-down list, select
NFQ-MGB
(default).
d.
Leave the default in the Color field.
3.
Set up the remaining targets as shown below:
4.
Click
Next
.
Note:
For all targets, leave blank the (Optional) Enter Gene Name field. You can search
for the gene/assay ID when you order your materials (see “Order Materials for the
Experiment” on page 131).
Target
Target Name
Reporter
Quencher
Color
2
EGR3
FAM
NFQ-MGB
(Use default color)
3
MAOB
FAM
NFQ-MGB
(Use default color)
4
OGDH
FAM
NFQ-MGB
(Use default color)
5
OSGEP
FAM
NFQ-MGB
(Use default color)
6
SERPING1
FAM
NFQ-MGB
(Use default color)
