ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Eliminating Outliers For any PCR, experimental error may cause some wells to amplify insufficiently or not
at all. These wells typically produce C T values that differ significantly from the average
for the associated replicate wells. If included in the absolute quantification
calculations, these outliers can potentially result in erroneous measurements.
6-12 Real-Time Analysis
Visualizing Outliers
To ensure precise absolute quantification, replicate groups must be carefully
scrutinized for outlying wells. The CT vs. Well Position view of the Amplification Plot
allows you to examine each set of replicate wells for outliers.
To visualize the replicate groups for outlying amplification:
Step Action
1 From the Plot drop-down list, select Ct vs. Well Position.
The SDS software displays the results data in a Well versus Threshold Cycle plot.
2 Verify the uniformity of each replicate population by comparing the groupings of
CT values for the wells that comprise the set.
Are outliers present? Then...
Yes a. Determine and record the well numbers of all
outlying wells.
b. Go to the next step.
No go to the next step.
3 Check for remaining detectors present on the plate for outliers:
a. Click the Detector drop-down list, and select another detector.
b. Repeat step 2 until each detector has been checked for outliers.
4 If outliers are present in your data, eliminate them as explained below. Otherwise,
go on to “Viewing Results” on page 6-13.
Eliminating Wells from the Analysis
IMPORTANT If one or more wells are removed from use before a plate document has been
run, the SDS software will not collect data for those wells.
If you identified any outliers in the previous procedure, you must eliminate them from
the standard curve calculation before viewing the results.
To eliminate outliers from the calculations:
Step Action
1 While pressing and holding the Ctrl key, click each well in the plate grid that you
identified as an outlier in the previous procedure.
The SDS software creates a black boarder around each cell as it is selected.
2 Click the Setup tab.
3 From the well inspector of the Setup tabbed page, uncheck the In Use check box.
4 From the Analysis menu, select Analyze.
5 Reapply the baseline and threshold values determined on page 6-10.