Applied Biosystems 7900HT User Manual

DRAFT
September 1, 2004 11:39 am, CH_Real-Time.fm
Overview
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-17
Algorithmic Manipulation of Raw Data
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.
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. At the same time, the software also
determines the contribution of each dye in the raw data using the multicomponent
algorithm.
Setting the
Threshold and
Calling Threshold
Cycles
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 page 6-19). The C
T
values computed
using the baseline and threshold data are the basis for generating gene expression
values.
Outlier Removal
For any PCR, experimental error may cause some wells to amplify insufficiently or
not at all. These wells typically produce C
T
values that differ significantly from the
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 page 6-19) or manually (see page 6-49).
Generating Gene
Expression
Values
The final stage in the analysis is the computation of gene expression values from the
C
T
data. The mathematical process for deriving relative quantification values is
briefly described on page D-8. For detailed information on the derivation of the
∆∆C
T
equation, relative quantification data transformations, or other aspects of
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
RISM 7700 Sequence Detection System User Bulletin #2: Relative
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
T
Equation, see:
– ABI P
RISM 7700 Sequence Detection System User Bulletin #2: Relative
Quantification Of Gene Expression (PN 4303859)

Applied Biosystems 7900HT Specifications

General

Brand	Applied Biosystems
Model	7900HT
Category	Laboratory Equipment
Language	English