Absolute Quantification estimates the quantity of an unknown sample by comparing to standards that have been programmed into the run. Quantities may be in terms of an actual copy number, mass, or concentration. The analysis makes use of Cq calling methods that derives Cq values from amplification curves measured in Automatic Quantification or Fit point Quantification.
Performing Absolute Quantification
To demonstrate absolute quantification, we will use an example data file. Open the “MyGo Pro 2 fold Dilution Series.ppf” file from the demo data folder. Now click the Profile tab, where we can see that a simple cycling profile has been run. All quantification analyses require a single cycling phase to be present in the profile. This can be a normal cycling phase or a touchdown phase.
Next we will review the samples and targets to see how they are configured for an absolute quantification analysis. Click on the Samples tab if not already selected.
Standard Target Quantities
We can see that the first 8 wells contain a 2-fold dilution series of a single target. The quantities of the target present are listed in the sixth column, titled Type, next to the name of the target. The “S” icon indicates that the target is present as a standard.
Estimating Unknown Target Quantities
By comparing the Cq values of unknown examples of the same target to the Cq values of these known standard quantities, the unknown quantities can be estimated. You may provide as many standards as required to cover the expected range of quantities of unknown targets. Providing multiple replicates of each quantity allows for errors to be estimated. In the example experiment, there are four replicates of each standard.
Scrolling down the Wells as Table display, assign FAM to C1-C8 as unknown. The quantification analysis will estimate quantities for these targets:
Select Analysis Type
Click the Analysis tab and add an Automatic Quantification analysis from the Select Analysis Type window. If not already present learn how to do this in the Selecting Analysis Type section. This is a quantification analysis performed using the Auto. Quant algorithm. Once the Cq values have been produced, further analysis proceeds in the same way for each algorithm. For now we will work with the Auto. Quant. algorithm, later sections will cover the details of using different Cq calling algorithms.
Results as Table
Click the Results as Table tab, if it is not already selected. This shows the common features present in the Results as Table layout, as shown in General Table Layout.
Samples as Plate
The Samples as Plate view shows the features described in Working with Plate Displays.
As you can see, the default settings will very often be suitable for analysing the data. Since the samples and targets are already set up, the analysis can proceed without any additional data.
Exporting Data from Results as Table
The data in the Results as Table display can be exported to a character separated value (.csv) file using the Export… button.
The graph has the common features of zooming, selecting curves, and exporting data. Note that the selection of wells is shared with the Results as Table, Samples as Plate and Cq Values as Plate displays. See Working with Graphs for details of the common graph features.
Click the Amplification tab in the top right pane, if it is not already selected. This shows a graph of the intensity (in relative fluorescence units, RFU) of the selected dye, against the cycle of amplification. There is one curve for each well that has a target labelled with the selected dye.
Graph Display Controls
By default, the selected graph curves are colored according to the Cq of the target associated with the selected dye in each well. Unselected wells are colored light grey. See earlier description of Cq Values as Plate for more details.
Coloring Amplification Curves by Sample
Click the Color by: drop down control, and select Sample:
The graph curves are now colored according to the sample in the wells - this coloring matches the Samples as Plate display.
Coloring Amplification Curves by Dye
In experiments where multiple dyes are used, the Dye: drop down control can be used to display different dyes. Since one dye can be used for multiple targets, all targets associated with the dye will be displayed. Alternatively, the graph can display all dyes present in the experiment, when Show all dyes? is selected
Notes, Errors and Warning Signs
Click on the Notes tab in the top right of the experiment area:
This area is used to display any information, warnings and/or errors generated by the analysis. For example, if any settings are invalid, an error will be displayed. Information might include hints to produce better results, etc. In this case, there are no notes generated by the analysis.
Samples as Plate
Please refer to Quantification Plate Displays to learn more about Samples as Plate when doing quantification analysis.