Analysis FAQ
Q: What is a Ct?
A: The most basic definition of a Ct is the first cycle in which there is a significant increase in fluorescence above the background.
Q: What is the difference between 2nd derivative and primary curve?
A: The 2nd derivative and primary curve are two ways to determine a cycle threshold value.
If the primary curve is used the cycle threshold reported is the intersection of the growth curve and the threshold.
If the 2nd derivative is selected the software determines the cycle threshold value by calculating the 2nd derivative of the growth curve. The software chooses the largest 2nd derivative peak for the cycle threshold (this is usually the inflection point of the growth curve).
Q: What is the difference between automatic and manual Threshold Settings?
A: If manual is selected, the user sets the fluorescence value used for the threshold. Manual mode gives the user more flexibility to evaluate background.
Automatic will always set threshold above background, however, it does not allow the user to choose the fluorescence value used for the threshold. Automatic mode calculates the threshold as the specified number of SDs above the background.
Q: I made changes to the analysis table but the graph did not update to reflect changes.
A: The Update Analysis button located at the bottom of the View Results screen must be selected to implement changes made to the Analysis Settings or Results Table.
Q: Is there a way to reduce the noise in my growth curves?
A: The boxcar average analysis setting averages data points, which reduces the noise displayed in the growth curve. The number of points (usually 2 or 3) is specified by the user. However, boxcar averaging can shift cycle threshold values by approximately one cycle.
Q: When background subtraction is “ON” I see drift of the baseline (positive or negative). Can this be corrected?
A: The background min and max cycle numbers can be adjusted to reduce the baseline drift.
Q: My negative samples are reporting Ct values in the Results Table.
A: If the threshold is set too low and baseline drift crosses the threshold a Ct may be reported. Increase the threshold value (manual mode) or the number of SDs used to calculate the automatic threshold.
Q: Can I make changes to the Analysis or Results table while a run is in progress?
A: Yes, changes can be made to the analysis settings at any time. Always click the Update Analysis button to implement changes made to the Results Table or Analysis Settings.
Note: If the Advance to Next Stage feature is used in software version 2.0, the analysis settings must be set up before the run has started.
Q: Can I use one standard curve for several runs?
A: Yes, a previous standard curve can be imported and saved with a run that contains only unknown samples. Click the Import Std Curve button at the bottom of the View Results screen to select a run from a list of all the valid standard curves in the database. (Chapter 5)
Q: Do I need to set-up the analysis settings before a run is started?
A: No, the analysis settings can be set up before, during or after a run. Each time a change is made to the analysis settings the Update Analysis button must be selected to implement the changes. To save changes made to the Analysis settings click the Save Run button.
Q: I selected sites as standards but my standard curve graph does not show any data.
A: The Update Analysis button must be selected after changes are made to the Results Table. Check that the standard values were entered in the correct column.