Applied Biosystems 7900HT - Page 355

A similar equation for the endogenous reference reaction is:
where:
Dividing X
T
by R
T
gives the following expression:
The exact values of X
T
and R
T
depend on a number of factors, including:
• 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
X
= E
R
= E,
or
where:
R
T
= threshold number of reference molecules
R
o
= initial number of reference molecules
E
R
= efficiency of reference amplification
C
T, R
= threshold cycle for reference amplification
K
R
= constant
X
N
=X
o
/R
o
, the normalized amount of target
∆C
T
=C
T, X
- C
T, R
, the difference in threshold cycles for target and reference
R
T
R
o
1( E
R
)+×
C
TR,
K
R
==
X
T
R
T
-------
X
o
1( E
X
)
C
TX,
+×
R
o
1( E
R
)
C
TR,
+×
---------------------------------------------
K
X
K
R
------- -
K===
X
o
R
o
------
1( E) K=
C
TX,
C–
TR,
+×
X
N
1( E)
C
T
∆
+× K=