Eliminating Outlying Amplification - Applied Biosystems 7900HT User Manual

Eliminating Outlying Amplification

Overview
Visualizing
Outliers
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
For any PCR, experimental error may cause some wells to amplify insufficiently or not
at all. These wells typically produce C
for the associated replicate wells. If included in the absolute or relative quantification
calculations, these outliers can potentially result in erroneous measurements.
To ensure precise relative quantification, replicate groups must be carefully
examined for outlying wells. The C
allows you to examine each set of replicate wells for outliers.
1. Select the Results tab.
2. If analyzing a Relative Quantification study, select a plate in the Plates field and
deselect all wells in the plate grid. Repeat for each plate in the Plates field.
When you are finished, the Amplification plot should not display any data.
3. If eliminating outliers from a:
Absolute Quantification Plate Document
Select all wells of the plate by clicking the upper-left corner of the plate grid.
Relative Quantification Multiple Plate Document
a. Select a plate in the Plates field to view.
b. Select all wells of the plate by clicking the upper-left corner of the plate grid.
4. In the Plot drop-down list, select Ct vs. Well Position.
5. In the Well versus Threshold Cycle Amplification plot, verify the uniformity of
each replicate population by comparing the groupings of C
that make up the set.
Are outliers present?
Yes
No
6. In the Detector drop-down list, select another detector, and repeat
through 5. Repeat until each detector has been screened for outliers.
7. If analyzing a Relative Quantification Multiple Plate Document, deselect the
wells of the current plate, then repeat
study.
8. Are outliers present on the plate?
Yes – If outliers are present for any detector, eliminate the outlying wells from
the analysis by omitting them as explained in
Analysis" on page
No – If no outliers are present, do one of the following:
• If analyzing an Absolute Quantification Plate Document, view the results of
the run displayed within the Standard Curve plot and Results table.
values that differ significantly from the average
T
vs. Well Position view of the Amplification plot
T
Then...
Record the well numbers of the outlying wells, then go to
the next step.
Go to the next step.
steps 3 through 6
6-50.
September 1, 2004 11:39 am, CH_Real-Time.fm
Eliminating Outlying Amplification
values for the wells
T
for every plate in the
"Eliminating Wells from the
DRAFT
steps 3
6-49