Reverse Calibration
(This feature is only available in GenEx Pro/Enterprise)
Given data for a standard curve, absolute quantities of unknown samples can be calculated by reverse calibration. Enter the data for the standard curve together with the unknown samples in the Data editor. The data should include one classification column that indicates the concentration for the standard curve samples and 0 (zero) for the test samples.
Define the samples with unknown concentrations as Test in the Data manager under the Data selection tab, and the samples with known concentrations as Training. Open the Regression tab among the analysis tabs in the top of the main window, and press the Reverse Calibration button to load the analysis into the Control panel.
Select the gene which measurements you want to use for calibration in the Gene (Y) drop-down list. Also select the classification column that contains concentration values to be used in the calibration in the Concentration (X) drop-down list. Options are available to find confidence intervals with confidence level of 90%, 95%, or 99%. The data set may contain a classification column indicating replicates which has identical numerical values at rows of replicate samples and different from all other samples. If the data have replicates, tick the Repeats exist check box and select the classification column that indicates the repeats. The analysis is based on input in logarithmic scale such as foldchanges and Cq values. If you have data on linear scale, such as copy numbers, tick the Apply log on (X) check box.
Press the Run button to see the results. A standard curve (blue line) is obtained by fitting a regression line to the samples with known concentrations (training samples). A confidence interval (red lines on either side of the standard curve) is calculated based on these data and on the confidence level that was selected in the Control panel. Test samples (samples with concentration value 0) are indicated with red dots at corresponding points on the standard curve.
A table shows the results for the standard curve analysis and the concentrations of the test samples. Each result comes with the estimated result as well as a lower and upper confidence limit. The upper section of the table gives the Slope and Intercept at zero concentration of the standard curve, and the calculated qPCR efficiency. The lower section gives estimated concentrations with confidence intervals of the test samples. The values in green columns are on logarithmic scale and the values in blue columns are on linear scale.
