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4.6 Analysis method: Quantification
This analysis method has two general approaches: either to determine the ‘absolute’ concentration
of an unknown sample by comparison of the quantification cycle (Cq) to a standard curve of known
concentrations or else a relative value determined by the comparison of Cqs. The latter approach
is useful in screening assays, for example, when an increase relative to a control sample provides
sufficient experimental information such that an ‘absolute’ concentration is not required. Both
approaches use Cq calculation as the basis of the quantification as reflected in the setup
procedure detailed below.
4.6.1 Assay requirements
•
Need at least one reporter.
•
Need at least two readings.
•
Need dye defined as PRD for ‘PRD correction’ options to be available.
•
Need at least two reporters for relative quantification methods.
This method allows baseline correction for amplification data followed by a quantification
calculation using one of two methods. Results can then be given as a quantification cycle (Cq), an
absolute concentration, if standards have been included, or relative concentration if more than one
reporter is being used.
4.6.2 Setup
The diagram below gives an overview of Quantification analysis.
•
In the Experiment Editor Analysis Selection box, highlight the stage name to which analysis
is to be applied and click Edit . The Analysis Wizard will launch.
•
Choose Quantification from the drop-down menu.
•
Assign a use for the dye(s) employed in the assay. Click
Next
.
•
The Quantification Wizard launches and leads the user through the quantification setup.
Raw data for
reporter with or
without PRD
correction
Baseline
correction
options
Quantification
options
Concentration
Report
Raw data for
reporter with or
without PRD
correction
Baseline
correction
options
Quantification
options
Concentration
Report