ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Calculating
Threshold Cycles
A-10 Theory of Operation
The ABI PRISM 7900HT Sequence Detection System creates quantifiable
relationships between test samples based on the number of cycles elapsed before
achieving detectable levels of fluorescence. Test samples containing a greater initial
template number cross the detection threshold at a lower cycle than samples
containing lower initial template. The SDS software uses a Threshold setting to define
the level of detectable fluorescence.
The threshold cycle (CT ) for a given amplification curve occurs at the point that the
fluorescent signal grows beyond the value of the threshold setting. The CT represents
a detection threshold for the 7900HT instrument and is dependent on two factors:
♦ Starting template copy number
♦ Efficiency of DNA amplification the PCR system
How the SDS Software Determines CTs To determine the CT for an amplification plot, the SDS software uses data collected
data from a predefined range of PCR cycles called the ‘baseline’ (the default baseline
occurs between cycles 3 and 15). First, the software calculates a mathematical trend
based on the baseline cycles’ Rn values to generate a baseline subtracted
amplification plot of ∆Rn versus cycle number. Next, an algorithm searches for the
point on the amplification plot at which the ∆Rn value crosses the threshold setting
(the default threshold setting is 0.2). The fractional cycle at which the intersection
occurs is defined as the threshold cycle (CT) for the plot.
Note It may be necessary to adjust the baseline and threshold settings to obtain accurate and
precise data. For further information on resetting the baseline and threshold settings, see
“Setting the Baseline and Threshold Values for the Run” on page 6-10.
R n
8.0
2.0
1.0
0.0
0
10
20
Cycle Number
30
40
1250
Threshold setting
Threshold cycle (C T )
for the 1250 copy well