Relative standard curve run
A relative standard curve runs determines the 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
) run
A comparative C
t
(
DD
C
t
) run determines the 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
Types of runs
1
32
QuantStudio
™
6 Pro Real-Time PCR System and QuantStudio
™
7 Pro Real-Time PCR System User Guide
