Applied Biosystems 7900HT User Manual page 353

Significance of
Threshold Cycles
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
Beginning with the equation describing the exponential amplification of the PCR:
–
n m
( )
X = X 1 + E
n m X
where:
number of target molecules at cycle n (so that n ≥ m)
X =
n
number of target molecules at cycle m (so that m " n)
X =
m
E = efficiency of target amplification (between 0–1)
X
n - m = number of cycles elapsed between cycle m and cycle n
Amplicons designed and optimized according to Applied Biosystems guidelines
(amplicon size <150 bp) have amplification efficiencies that approach 100 percent.
Therefore E =1 so that:
X
–
n m
( )
X = X 1 + 1
n m
n m –
2 ( )
= X
m
To define the significance in amplified product of one thermal cycle, set n - m = 1 so
that:
1
2 ( )
X = X
1 0
= 2X
0
Therefore, each cycle in the PCR reaction corresponds to a two-fold increase in
product. Likewise, a change in threshold cycle number of one must equate to a
two-fold difference in initial template concentration.
DRAFT
September 1, 2004 11:39 am, App_Theory.fm
Real-Time Data Analysis
D-7