Qiagen QIAcuity 911000 User Manual page 211

2D Scatterplot tab for absolute quantification
The 2D Scatterplot tab shows a 2D Scatterplot view that displays the fluorescence intensity from two
selected channels or targets, allowing for a comparison and evaluation of the fluorescence from
two sources.
To create a 2D Scatterplot, assign targets or channels to the x-axis and y-axis using the drop-down
menus. When 2 of the selected sources for analysis are assigned, the 2D Scatterplot view is
displayed. The axes represent the value of fluorescence intensity. The ranges of the axis are aligned
with the maximal values presented from 0 to maximal fluorescence intensity of selected channel
measured. The user can change the range of the x-axis and the y-axis by modifying the upper value
of the fluorescence intensity on the x-axis and the y-axis. For this, the user adjusts the value in the
field to Max value for the x-axis (RFU) or Max value for the y-axis [RFU] and press Save. Resetting
the x-axis and the y-axis to the default range is provided by using the Back to Default button.
The Application also presents number of double positives (++) and double negatives (−−), and
number of partitions that are positives on one channel and negative on the other (+−, −+). To view
updated results after changing threshold, click the Recalculate button.
To view an additional toolbar that enables you to perform actions related to the 2D Scatterplot
view, such as downloading the plot, hold the pointer over the diagram. For more details about the
toolbar, refer to Diagram options.
The Common threshold field for each axis shows the Common threshold value for each selected
well of the fluorescence intensity that is used to distinguish between positive and negative partitions.
If only one source well is selected, the values of the thresholds for the targets or channels on each
axis are shown in the Common threshold fields and on the graph in the form of red lines. If multiple
source wells are defined and their automatically calculated threshold values are different, a
Common threshold value is initially not shown.
Setting the Common thresholds for all selected wells and each axis (channel)
The 2D Scatterplot view provides an option to set a common threshold value for the x-axis and y-
axis for all associated wells. There are two ways to change the threshold. The first one involves
pointing to the chart, which triggers the appearance of two perpendicular dotted lines. Once the
dotted lines are in the appropriate spot, click the chart. The lines become solid, and the threshold
value is updated, and shown in the Threshold field. To change the value again using this method,
click one of the red lines, and drag it to the appropriate spot. If needed, repeat this step with the
second line. In addition to moving the lines, you can also directly edit the value in the Common
threshold field of the associated axis. Click the Recalculate button to trigger the re-analysis of data.
QIAcuity User Manual 06/2022 211