Overview - Applied Biosystems 7900HT User Manual

Chapter 6 Analyzing Real-Time Data

Overview

Relative
Quantification on
the 7900HT
Instrument
Employing the
5´ Nuclease
Assay
6-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
September 1, 2004 11:39 am, CH_Real-Time.fm
The Applied Biosystems 7900HT Fast Real-Time PCR System supports real-time
relative quantification of nucleic acids using the Comparative C
Comparative C Method uses the following arithmetic formula to achieve results for
T
relative quantification:
∆ ∆
–
C
2
T
where:
∆∆C
= The normalized signal level in a sample relative to the normalized signal level
T
in the corresponding calibrator sample.
The derivation of the above equation is explained in ABI P
Note:
Detection System User Bulletin #2: Relative Quantification of Gene Expression
(PN 4303859).
The normalized signal levels discussed above refer to the signals generated by
Note:
the amplification of the target sequence in the unknown and calibrator samples which
is normalized to the signal generated by the amplification of the endogenous control.
Note: The SDS software does not support the Standard Curve Method of relative
quantification.
Relative quantification on the 7900HT instrument is accomplished through the use of
the polymerase chain reaction and the fluorogenic 5´ nuclease assay. After careful
selection of an endogenous control, probe and primer sets are designed for both the
target and control sequences. After the assays are verified, unknown samples are
loaded onto an optical plate containing master mix and 5'-nuclease assays targeting a
specific nucleic acid sequence. The reaction plate is then run on a 7900HT
instrument that is configured for real-time analysis.
During the run, the instrument records the emission resulting from the cleavage of
®
TaqMan probes in the presence of the target sequence. After the run, the SDS
software processes the raw fluorescence data to produce threshold cycle (C
for each sample. The SDS software computes relative quantities from the C
of the calibrator sample and the data from the unknown samples within the gene
expression profile.
Note: See Appendix D, "Theory of Operation,"
fluorogenic 5´ nuclease assay, real-time data collection, or the mathematical
transformations of sequence detection data.
DRAFT
method. The
T
7700 Sequence
RISM
for more information on the
) values
T
values
T