Applied Biosystems 7900HT User Manual page 355

A similar equation for the endogenous reference reaction is:
C
× 1 ( )
,
= +
R R E T R
T o R
where:
R = threshold number of reference molecules
T
R = initial number of reference molecules
o
E = efficiency of reference amplification
R
C = threshold cycle for reference amplification
T, R
K = constant
R
Dividing X by R gives the following expression:
T T
C
× 1 ( ) T X
X + E
X
o X
T
---------------------------------------------
------ - =
C
R
× 1 ( ) T R
T
R + E
o R
The exact values of X
T
• Reporter dye used in the probe
• Sequence context effects on the fluorescence properties of the probe
• Efficiency of probe cleavage
• Purity of the probe
• Setting of the fluorescence threshold
Therefore, the constant K does not have to be equal to one.
Assuming efficiencies of the target and the reference are equivalent:
E = E = E,
X R
X
C – C
,
,
o T X
× 1 ( E ) T R
------
+
R
o
or
∆
C
× 1 ( E ) T
X + = K
N
where:
X = X /R , the normalized amount of target
N o o
∆C
= C - C , the difference in threshold cycles for target and reference
T T, X T, R
=
K
R
,
K
X
------- -
= = K
K
,
R
and R depend on a number of factors, including:
T
= K