abi 7500 - Set Up the Targets

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)