This Information Applies To: Agilent AriaMx real-time PCR system
After a run, the Cq values of wells change, depending which wells are selected.
It is normal and expected behavior for e Cq values of wells change, depending which wells are selected. The reason for this is that the AriaMx software uses a background-based algorithm to calculate the threshold for Cq detection. All wells within the selection that have the same target (either a user-defined target, or just the detection channel) will be used to calculate the threshold. If the selection is changed, the calculated threshold will most likely change, along with the Cq values. So that all wells have the same threshold all wells of the same assay should be analyzed together. If multiple targets are detected using the same channel, it's necessary to assign targets.
Tip: Targets are assigned on the Plate Setup tab, in the Properties menu on the right side of the screen.
For the background-based threshold calculation, the software takes the background signal for all the selected wells within the defined cycle range and multiplies the background noise with the provided sigma multiplier. Both parameters are set within the software.
The default values are displayed in Figure 1.
Figure 1. Background-based threshold default values
Table 1 gives an example of Cq value change depending on cell selection using the AriaMx sample experiment Quantitative PCR DNA Binding Dye with Melt [Sample]. This file, along with other sample experiments, is automatically saved to folder C:\Users\Public\Public Documents\Agilent Aria\Sample Experiments when the AriaMx software is installed. When more wells are selected, the variability in the fluorescent baseline also increases, i.e. the background noise increases, which increases the calculated threshold, resulting higher Cq values.
|Type of Selection||Cq Value for the well C2|
|Single Well selected - C 2 (Figure 2)||19.40|
|Single Column selected - Column 2 (Figure 3)||19.43|
|Whole plate selected (Figure 4)||19.58|
Table 1. Variable Cq values for well C2 in the sample data file Quantitative PCR DNA Binding Dye with Melt [Sample].
Figure 2. Variable Cq values for well C2: single well selected. Well C2 is highlighted with a red square.
Figure 3. Variable Cq values for well C2: single column selected. Well C2 is highlighted with a red square.
Figure 4. Variable Cq values for well C2: whole plate selected. Well C2 is highlighted with a red square.
Caution: If not all wells of the same target are selected when the analysis is performed, incorrectly low Cq values or false positive detection is possible.
|Learn how to effectively operate your Agilent AriaMx:
AriaMx Real-Time PCR System Setup and User Guide on Agilent.com
Aria Real-Time PCR Software User Manual on Agilent.com
For Research Use Only. Not for use in diagnostic procedures.