Algorithmic Manipulation Of Raw Data - Applied Biosystems 7900HT User Manual

Algorithmic Manipulation of Raw Data

Multicomponenting
Setting the
Threshold and
Calling Threshold
Cycles
Outlier Removal
Generating Gene
Expression
Values
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
The SDS software can analyze raw data immediately after a relative quantification
run is complete. The term "raw data" refers to the spectral data between 500 nm to
660 nm collected by the software during the real-time run. 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.
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. At the same time, the software also
determines the contribution of each dye in the raw data using the multicomponent
algorithm.
After multicomponenting, the baseline and threshold values must be set for each
detector in the study. The software allows you to set the baseline and threshold values
manually (see page 6-49) or automatically (see
using the baseline and threshold data are the basis for generating gene expression
values.
For any PCR, experimental error may cause some wells to amplify insufficiently or
not at all. These wells typically produce C
average for the associated replicate wells. If included in the relative quantification
calculations, these outliers can potentially result in erroneous measurements. To
ensure precise relative quantification, replicate groups must be carefully scrutinized
for outlying wells before the analysis. The software provides options for removing
outliers automatically (see
The final stage in the analysis is the computation of gene expression values from the
C data. The mathematical process for deriving relative quantification values is
T
briefly described on page
∆∆C
equation, relative quantification data transformations, or other aspects of
T
5´ nuclease chemistry in relation to relative quantification, Applied Biosystems
recommends the following references:
• For information on performing relative quantification using
5´ nuclease chemistry on 7900HT instruments, see:
– ABI P 7700 Sequence Detection System User Bulletin #2: Relative
RISM
Quantification Of Gene Expression (PN 4303859)
– RQ Manager Software User Guide(PN 4351670)
– The bibliography of this document for a list of technical papers and research.
• For information on the derivation of the ∆∆C
– ABI P 7700 Sequence Detection System User Bulletin #2: Relative
RISM
Quantification Of Gene Expression (PN 4303859)
page
values that differ significantly from the
T
page 6-19) or manually (see
D-8. For detailed information on the derivation of the
DRAFT
September 1, 2004 11:39 am, CH_Real-Time.fm
6-19). The C values computed
T
page 6-49).
Equation, see:
T
Overview
6-17