Comparative C Method Of Relative Quantification - Applied Biosystems 7900HT User Manual

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