R-Biopharm Rida Cycler, Руководство пользователя

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RIDA
®
CYCLER
Version 1.0
|
User manual
58
Threshold start: you can avoid interfering parts at the start of the baseline-corrected real time
curve by moving the threshold start position. This can be achieved by either entering the value
in the Threshold start text box or moving your mouse left or right clicked on the green box at
the start of the threshold line.
It is not recommended to analyze two different assays using the same
cycle threshold or W-o-L. Different assays will have different efficiencies
that can affect the performance of the analysis algorithms..
Fluorescence Cutoff: This parameter will exclude samples that fall below a user defined
percentage of the maximum fluorescence change. Small changes in fluorescence can interfere
with the determination of Cq values by changing the W-o-L. Some of these small changes can
be due to probe self-hydrolysis and cross talk between dyes. The default level is set to 5 %.
Auto Exclusion: If the sample is excluded its Cq value is not determined and it is not included
in the window of linearity calculation. Depending on the exclusion setting a sample may be
excluded for one or more of the following reasons:
No amplification: The filtered fluorescence trace for the sample does not show sufficient
amplification. The change in fluorescence for the sample must increase by 7 times. The
criterion is taken from 'Amplification efficiency: linking baseline and bias in the analysis of
quantitative PCR data' Nucleic Acids Research, 2009, Vol. 37, No. 6 e45, in the caption for
Figure 3. To be included the sample fluorescence must satisfy the following condition
f_first-
f_min≥7×(f_max-f_min)
Where;
f_min is the smallest filtered fluorescence value;
f_first is the first filtered fluorescence value different to f_min;
f_max is the maximum filtered fluorescence value.
Fluorescence Cutoff: The largest value for the filtered fluorescence trace for the sample does
not exceed the fluorescence cut-off level and fluorescence cut-off is enabled.