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

Algorithmic
Manipulation of
Raw Data
The SDS software can analyze raw data immediately upon completion of absolute
quantification run. The term raw data refers to the spectral data between 500 nm to
660 nm collected by the Automation Controller Software during the plate-read. During
the analysis, the software automatically applies several mathematical transformations
to the raw data to generate a more direct measure of the relationship between the
spectral changes in the unknown samples.
Multicomponenting
The first mathematical transformation involves the conversion of the raw data,
expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye components
using the extracted pure dye standards. After the dye components have been
identified, the software determines the contribution of each dye in the raw data using
the multicomponent algorithm. See “Multicomponenting” on page A-5 for a complete
description.
Setting the Threshold and Calling C Ts
After multicomponenting, the baseline and threshold values must be set for the run
(see “Kinetic Analysis/ Quantitative PCR” on page A-7 for more information). The
results of the experiment can be visualized in the Standard Curve graph of the Results
tab. The graph consists of a scatter plot of standard and unknown samples graphed
on a linear-scale plot of Threshold Cycle (C T) versus Starting copy number.
Real-Time Analysis 6-5