Within the acquisition type of qPCR amplification, relative quantification of the results is supported by FastFinder Analysis. FastFinder Analysis implements the Pfaffl method for relative quantification, which takes differences in the PCR efficiencies for the reference and target genes into account.
The Assay Plugin needs to be correctly configured to support this type of quantification.
The implementation and visualisation of the relative quantification (results) will be highlighted here.
Lab Configuration
A new sample type is introduced, i.e. Reference sample which is required to perform relative quantification.
In the PCR tab, the settings for the relative quantification are shown. In the PCR tab of the Assay Plugin (in Lab configuration), the configuration for relative quantification can be found.
The efficiency of the target and/or reference gene can be adjusted by (Admin) users (if this value is made lab-depending in the Assay Plugin).
Analysis
PCR set up
When assigning the correct Assay Plugin or making changes to the existing plate setup, the reference sample type (when configured in the Assay Plugin) can be assigned to the desired well(s).
Summary
In the Summary tab, the Relative Quantification Results are shown, similar as for the Control Results. Here, the result for the reference sample is provided, as is the validity.
When the reference sample is valid, the relative quantification of the regular samples is calculated and the results are shown in the Sample Results, displayed as 'RQ' next to the individual target result and Cq value. This value can also be exported from FastFinder, if needed (for more information, see Settings - Analysis results CSV).
Details
In the Details, an overview of the results per target is visualized, including the result for the reference sample itself. For regular samples, the relative quantification result is added.
Graphical layout
The results of the relative quantification are also visualized in a graphical manner, and can be found under the plate layout. The settings are very similar to those of the standard graphical layout (for more information, see Analysis - Graphical layout).
The curve color of the graph can be organised either by sample or by target (changeable via the graph settings (gear wheel icon in the top right corner)).