Experiment types
Purpose
Description
Standard curve experiment
Determines absolute
target quantity in
samples.
1.
The software measures amplification of the target in a standard dilution series and in
test samples.
2.
The software generates a standard curve using data from the standard dilution series.
3.
The software uses the standard curve to interpolate the absolute quantity of target in
the test samples.
Relative standard curve experiment
Determines relative
target quantity in
samples.
1.
The software measures amplification of the target of interest and of an endogenous
control target in a standard dilution series, in a reference (calibrator) sample, and in test
samples.
The endogenous control is a target that is expressed equally in all samples;
examples of endogenous controls are β‐actin, GAPDH, and 18S ribosomal RNA. The
software can algorithmically incorporate multiple endogenous control targets in relative
quantification calculations.
The reference sample is used as the basis for relative quantification results (or
1× sample). For example, in a study of drug effects on gene expression, an untreated
control is an appropriate reference sample.
2.
The software generates standard curves for the target of interest and the endogenous
control using data from the corresponding standard dilution series.
3.
The software uses the standard curves to interpolate the quantities of the target of
interest and the endogenous control in each sample. The target quantity in each
sample is then normalized to the sample's endogenous control quantity.
4.
To determine the relative quantity of the target in test samples, the software divides
the normalized target quantity in the sample by the normalized target quantity in the
reference sample.
Comparative C
t
(
DD
C
t
) experiment
Determines relative
target quantity in
samples.
1.
The software measures amplification of the target of interest and of an endogenous
control target in a reference (calibrator) sample and in test samples.
The endogenous control is a target that is expressed equally in all samples;
examples of endogenous controls are β‐actin, GAPDH, and 18S ribosomal RNA. The
software can algorithmically incorporate multiple endogenous control targets in relative
quantification calculations.
The reference sample is used as the basis for relative quantification results (or
1× sample). For example, in a study of drug effects on gene expression, an untreated
control is an appropriate reference sample.
2.
The measurements for the target of interest are normalized to the endogenous control.
3.
To determine the relative quantity of the target in test samples, the software compares
the normalized ΔC
q
(ΔC
t
or ΔC
rt
) for the sample to the normalized ΔC
q
(ΔC
t
or ΔC
rt
) for
the reference sample.
Chapter 1
Product information
Experiment types
1
QuantStudio
™
1 Plus Real-Time PCR System Installation, Use, and Maintenance Guide
19
