abi 7500 - Page 52

Chapter 2 Design the Relative Standard Curve Experiment
Set Up the Targets
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
28
Notes
Design
Guidelines
When you design your own relative standard curve experiment:
• Select the Set Up Standards check box. Applied Biosystems recommends that you
set up a standard curve for each target assay in the reaction plate.
• Identify each target assay with a unique name and color. You can enter up to
100 characters in the Target Name field.
• Select an endogenous control for each sample. The endogenous control is a target
that is in all samples under investigation. It should be expressed equally in all
sample types, regardless of treatment or tissue origin (examples of endogenous
controls are β-actin, GAPDH, and 18S ribosomal RNA [18S rRNA]). The
endogenous control is used to normalize the PCR results; the endogenous control
corrects for variable sample mass, nucleic acid extraction efficiency, reverse
transcription efficiency, and pipette calibration errors. Note that:
– Each sample type (for example, each tissue in a study comparing multiple tissues)
requires an endogenous control.
– If samples are on multiple plates, each reaction plate must have an endogenous
control. Additionally, every plate must include an endogenous control for every
sample type on the reaction plate.
• Select the reporter dye used in the target assay. In the Methods and Materials screen
on page 24, if you selected:
–TaqMan
®
Reagents – Select the dye attached to the 5′ end of the TaqMan probe.
–SYBR
®
Green Reagents – Select SYBR.
• Select the quencher used in the target assay. In the Methods and Materials screen on
page 24, if you selected:
–TaqMan
®
Reagents – Select the quencher attached to the 3′ end of the TaqMan
probe.
–SYBR
®
Green Reagents – Select None.
For More
Information
For more information on:
• Completing the Targets screen – Open the 7500 Software Help by clicking or
pressing F1.
• Selecting an endogenous control – See the Application Note Using TaqMan
®
Endogenous Control Assays to Select an Endogenous Control for Experimental
Studies.