Chapter 1
Get Started
About Relative Standard Curve and Comparative C
T
Experiments
7
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
About Relative Standard Curve and Comparative C
T
Experiments
Real-Time PCR
Experiments
Relative standard curve and comparative C
T
(
∆∆
C
T
) experiments are real-time PCR
experiments. In real-time PCR experiments:
• The instrument monitors the progress of the PCR as it occurs.
• Data are collected throughout the PCR process.
• Reactions are characterized by the point in time during cycling when amplification
of a target is first detected.
Note:
In this guide, the term
experiment
refers to the entire process of performing a run
using the 7500/7500 Fast system, including setup, run, and analysis.
About Relative
Standard Curve
Experiments
The relative standard curve method is used to determine relative target quantity in samples.
With the relative standard curve method, the 7500 software measures amplification of the
target and of the endogenous control in samples, in a reference sample, and in a standard
dilution series. Measurements are normalized using the endogenous control. Data from the
standard dilution series are used to generate the standard curve. Using the standard curve,
the software interpolates target quantity in the samples and in the reference sample. The
software determines the relative quantity of target in each sample by comparing target
quantity in each sample to target quantity in the reference sample.
Relative standard curve 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 relative
standard curve 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
.
•
Standard
– A sample that contains known standard quantities; used in quantitation
experiments to generate standard curves.
•
Standard dilution series
– A set of standards containing a range of known
quantities. The standard dilution series is prepared by serially diluting standards.
•
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.
