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

Normalization of
Reporter Signals
A-6 Theory of Operation
The software uses the pure dye spectra, generated as part of instrument calibration
(see page 7-17), to solve for coefficients a, b, and c in the following equation:
Measured spectrum = a( FAM)
+ bVIC ( ) + cROX ( ) + d( Background)
+ MSE
where the coefficients a, b, and c represent each dye component’s contribution to the
composite spectrum.
Note The example calculation above assumes that pure dye components exist for three dyes
(FAM,VIC,andROX) and for the instrument background.
After solving for a, b, c, and d, the algorithm calculates the mean squared error (MSE),
which measures how closely the collective multicomponent spectrum conforms to the
raw spectra. The figure below shows a typical display of the contribution of each
component spectra for one well.
Fluorescence
Temperature
12000
8000
4000
0
93
83
73
63
53
Multicomponent Plot
0:10 0:30 0:50
Time
6978 1:10 1:30
FAM component
VIC component
ROX component
Background
component
MSE
While multicomponenting illustrates absolute change in emission intensity, the SDS
software displays cycle-by-cycle changes in normalized reporter signal (R n ). The SDS
software normalizes each reporter signal by dividing it by the fluorescent signal of the
passive reference dye. Because the passive reference is a component of the PCR
master mix, it is present at the same concentration in all wells of the plate. By
normalizing the data using the passive reference, the software can account for minor
variations in signal strength caused by pipetting inaccuracies and make better
well-to-well comparisons of reporter dye signal.
Note For the example above, the resulting data from the normalizing is displayed as FAM R n
and VIC R n .