The software uses quantifiers to allow for correction of efficiency of different PCR targets, and to derive absolute and relative quantities. The software can allow for all combinations of internal and external standards. Internal standards may be omitted, included for one quantity, or included for multiple quantities. Where efficiency cannot be derived from internal standards, it can be specified.
The following sections show how the quantifier system works in each case.
Full internal standards
When internal standards for two or more distinct quantities are present in an experiment, these internal standards are used to produce a standards curve which is used for both efficiency correction of Cq values, and target quantification. In this case, the internal standards can also be exported and then imported for use as external standards in other experiments. Note that standards for at least 3 distinct quantities are required for meaningful error and r-squared statistics to be generated.
The standards are shown on a graph of Cq against Log (base 10) of quantity. Each red point represents a Cq call. The blue line is the fitted linear regression curve used to convert Cq values into quantities.
Below the standards curve graph is the equation of this linear regression curve.
The r-squared value of the correlation is also displayed, as is the efficiency of amplification derived from the standards curve slope. An E value of 2 indicates ideal amplification, with a doubling per cycle. A value of 1 would indicate no amplification per cycle, etc. The Error value gives a measure of the average size of the difference between Cq values and the linear regression curve. Specifically, this is the root mean squared error of the linear fit to the standards.
At the bottom of the Quantifiers pane there is an Export Standards button.
Clicking the Export Standards button allows you to save the quantifier to a PCR Standards Quantifier (.psf) file. This can then be imported into any other experiment using the Import button. This allows results from one Experiment to be quantified based on standards in another Experiment.
No Internal Standards
When no internal standards are present, efficiency correction and relative quantification can be performed, but absolute quantification cannot be performed. There are two possibilities in this case, depending on whether external standards are imported.
No Internal Standards, with no External Standards
In the event that no external standards are imported, you can specify the target efficiency directly, and this efficiency is used to produce efficiency corrected Cq values, but not to quantify targets. In the example below, notice that all samples are unknown and therefore, no quantities have been derived. The efficiency has been set to 2, corresponding to a doubling of quantity per cycle of PCR.
No Internal Standards, with External Standards
To import external standards to the experiment click the Import Standards button at the bottom of the Quantifiers pane. These import standards will be used to specify the target efficiency, and this efficiency is in turn used to produce efficiency corrected Cq values, but not to quantify targets. A graph is shown, displaying the external standards curve, and the curve equation, efficiency and error/r-squared statistics are also shown:
Single Internal Standard
When internal standards for exactly one distinct quantity are present, these standards can be used with an efficiency value to perform efficiency correction, relative quantification and absolute quantification. There are two possibilities in this case, depending on whether external standards are imported.
Single Internal Standard with no External Standards
In the event that no external standards are imported, the user can specify the target efficiency directly, and this will be used for both efficiency corrected Cq values and quantification of targets. To create one internal standard select the Sample tab and apply one standard to all replicates of a single dilution as shown below:
Now select the Auto. Quant. module under the Analysis tab. Under the Quantifiers tab a graph displays one point, representing a single standard, with an illustrative line at the correct gradient for the specified efficiency. Calculated quantities are displayed in the results table.
Single Internal Standard with External Standards
External standards can be imported using the Import Standards button. These external standards will specify the target efficiency and this is used together with the single standard internal standard to produce a standards curve. This can be used for both efficiency corrected Cq values and quantification of targets.
Below is the default Quantifier view when external standards have been imported. Note that only the internal standards and gradient for imported efficiency are shown.
When “Graph Imported Stds.” has been checked the display is modified as shown below. The imported standards are displayed using a dashed line and hollow boxes, as indicated in the key below graph. Internal standards and efficiency are still shown with a solid line and filled boxes. Note that the solid internal standards curve is at the same gradient as the imported dashed standards curve, since it uses the same efficiency value.
Adding and Removing External Standards
Whenever imported external standards are in use, a button is displayed labelled Remove Imported Stds. which can be used to remove the standards. When this is clicked, external standards are removed and the button disappears. Whenever imported external standards could be used, a button is displayed labelled Import Standards.