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4.5
Analysis method: Baseline correction
This method allows the user to adjust the data for any background fluorescence. This approach
looks at the signal in the early cycles of the PCR and then averages out the early noise and
subtracts it from subsequent readings. As with passive reference dye (PRD) correction detailed
above, baseline correction can be useful for correcting the data prior to performing an analysis and
so can help to increase the accuracy of the assay.
4.5.1 Assay requirements
•
Need at least one reporter.
•
Need at least two readings.
•
Need a dye defined as a PRD for PRD correction options to be available.
4.5.2 Setup
The diagram below gives an overview of the different baseline correction methods.
•
Select
Baseline
from the drop-down menu in the Analysis Wizard Selection box. Assign the
dye usage and click
Next
. The Baseline Correction Wizard will launch.
4.5.2.1
PRD correction
If a PRD was assigned in the dye usage screen, the first screen of the wizard will ask if the data is
to be PRD corrected.
Raw data for Reporter with
or without PRD correction
None:
No baseline
correction
Arithmetic 1:
1.
For each well, calculate
average of readings in
range x to y.
2.
Subtract average from
all readings
Arithmetic 2:
1.
For each well, calculate
average of n lowest
readings.
2.
Subtract average from all
readings.
Proportional:
1.
For each well, calculate
average of n1 lowest
readings.
2.
Calculate average of n2
highest readings
(maximum).
3.
Calculate each reading as
% of the maximum.
SELECT BASELINE CORRECTION METHOD
Software plots fluorescence vs.
cycle no. using corrected data
Raw data for Reporter with
or without PRD correction
None:
No baseline
correction
Arithmetic 1:
1.
For each well, calculate
average of readings in
range x to y.
2.
Subtract average from
all readings
Arithmetic 2:
1.
For each well, calculate
average of n lowest
readings.
2.
Subtract average from all
readings.
Proportional:
1.
For each well, calculate
average of n1 lowest
readings.
2.
Calculate average of n2
highest readings
(maximum).
3.
Calculate each reading as
% of the maximum.
SELECT BASELINE CORRECTION METHOD
Software plots fluorescence vs.
cycle no. using corrected data
