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
QuantStudio
™
6 Pro and 7 Pro Real-Time PCR Systems User Guide
31
