Chapter 1
Get Started
About Relative Standard Curve and Comparative C
T
Experiments
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
8
Notes
•
Negative Controls
– Wells that contain water or buffer instead of sample template.
No amplification of the target should occur in negative control wells.
About
Comparative C
T
Experiments
The comparative C
T
(
∆∆
C
T
) method is used to determine the relative target quantity in
samples. With the comparative C
T
method, the 7500 software measures amplification of
the target and of the endogenous control in samples and in a reference sample.
Measurements are normalized using the endogenous control. The software determines
the relative quantity of target in each sample by comparing normalized target quantity in
each sample to normalized target quantity in the reference sample.
Comparative C
T
experiments are commonly used to:
• Compare expression levels of a gene in different tissues.
• Compare expression levels of a gene in a treated sample vs. an untreated sample.
• Compare expression levels of wild-type alleles with those of mutated alleles.
Components
The following components are required when setting up PCR reactions for comparative
C
T
experiments:
•
Sample
– The sample in which the quantity of the target is unknown.
•
Reference sample
– The sample used as the basis for relative quantitation results.
For example, in a study of drug effects on gene expression, an untreated control
would be an appropriate reference sample. Also called
calibrator
.
•
Endogenous control
– A target or gene that should be expressed at similar levels in
all samples you are testing. The endogenous control is used to normalize
fluorescence for the target you are quantifying. Housekeeping genes can be used as
endogenous controls.
•
Replicates
– The total number of identical reactions containing identical samples,
components, and volumes.
•
Negative Controls
– Wells that contain water or buffer instead of sample template.
No amplification of the target should occur in negative control wells.
