Chapter 1
Get Started
About Relative Standard Curve and Comparative C
T
Experiments
9
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
Relative Standard
Curve vs.
Comparative C
T
Experiments
Consider the following when choosing between relative standard curve and comparative
C
T
experiments:
Comparative C
T
(
∆∆
C
T
)
Experiment
Relative Standard Curve
Experiment
Description
Uses arithmetic formulas to determine
the change in expression of a target in a
sample relative to the same target in a
reference sample. Best for high-
throughput measurements of relative
gene expression of many genes in many
samples.
Uses a standard curve to determine the
change in expression of a target in a
sample relative to the same target in a
reference sample. Best for assays that
have suboptimal PCR efficiency.
Advantage
• Relative levels of target in samples
can be determined without the use
of a standard curve, if the PCR
efficiencies of the target and
endogenous control are relatively
equivalent.
• Reduced reagent usage.
• More space available in the reaction
plate.
Requires the least amount of validation
because the PCR efficiencies of the
target and endogenous control do not
need to be equivalent.
Limitation
• Suboptimal (low PCR efficiency)
assays may produce inaccurate
results.
• Before you use the comparative C
T
method, Applied Biosystems
recommends that you determine that
the PCR efficiencies for the target
assay and the endogenous control
assay are approximately equal.
A standard curve must be constructed
for each target, which requires more
reagents and more space in the
reaction plate.
