Chapter 2
Design the Relative Standard Curve Experiment
Set Up the Standards
29
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 Standards
In the Standards screen, enter the number of points and replicates for all standard curves
in the reaction plate. For each standard curve, enter the starting quantity and select the
serial factor.
About the
Example
Experiment
In the relative standard curve example experiment:
• One standard curve is set up for the target (c-myc). The standard used for the
standard dilution series is a cDNA sample of known quantity prepared from RNA
isolated from lung tissue.
• One standard curve is set up for the endogenous control (GAPDH). The standard
used for the standard dilution series is a cDNA sample of known quantity prepared
from RNA isolated from lung tissue.
• For each standard curve:
– Five points are used in the standard curve.
– Three replicates are used for each point. Replicates are identical reactions,
containing identical reaction components and volumes.
– The starting quantity is 200 ng, and the serial factor is 1:10.
Complete the
Standards Screen
1.
In the Standards screen, click the
How many points do you need for each
standard curve?
field, then enter
5
.
2.
Click the
How many replicates do you need for each point?
field, then enter
3
.
3.
Define the range of standard quantities for the c-myc assay:
a.
Click the
Enter Starting Quantity
field, then enter
200.0
.
b.
In the Select Serial Factor drop-down list, select
1:10
.
4.
Define the range of standard quantities for the GAPDH assay:
a.
Click the
Enter Starting Quantity
field, then enter
200.0
.
b.
In the Select Serial Factor drop-down list, select
1:10
.
5.
Review the Standard Curve Preview pane for each assay. The standard curves have
the following points: 200, 20, 2, 0.2, and 0.02.
6.
Click
Next
.
