Applied Biosystems 7900HT - Comparative C Method of Relative Quantification

DRAFT
September 1, 2004 11:39 am, App_Theory.fm
Appendix D Theory of Operation
D-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
Comparative C
T
Method of Relative Quantification
Arithmetic
Formula
The Comparative C
T
Method is similar to the Standard Curve Method. However, it
uses arithmetic formulas to achieve the same results for relative quantification. The
amount of target, normalized to an endogenous reference and relative to a calibrator,
is given by:
Derivation of the
Formula
The equation that describes the exponential amplification of PCR is:
where:
The threshold cycle (C
T
) indicates the fractional cycle number at which the amount
of amplified target reaches a fixed threshold.
Thus,
where:
2
C
T
∆∆–
X
n
= number of target molecules at cycle n
X
o
= initial number of target molecules
E
X
= efficiency of target amplification
n
= number of cycles
X
T
= threshold number of target molecules
C
T,X
= threshold cycle for target amplification
K
X
= constant
X
n
X
o
1( E
X
)+×
n
=
X
T
X
o
1( E
X
)+×
C
TX,
K
X
==