Rotor-Gene Q MDx CE User Manual 02/2022
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Standard normalization simply takes the first 5 cycles and uses these as an indicator of the
background level of each sample. All data points for the sample are then divided by this value to
normalize the data. This can be inaccurate because for some samples the background level over
the first 5 cycles may not be indicative of the background level just prior to amplification. In contrast,
dynamic tube normalization uses the second derivative of each sample trace to determine a takeoff
point for each sample. The background level is then averaged from cycle 1 up to this takeoff cycle
number for each sample. This gives the most precise quantitation results.
Note that for some data sets, background fluorescence is not consistent during the cycles before
amplification commences. In these cases, it may be necessary to deselect dynamic tube
normalization by clicking on
Dynamic Tube
because it could result in less precise quantitation.
Noise slope correction
The background fluorescence (Fl) of a sample should ideally remain constant before amplification.
However, sometimes the Fl shows a gradual increase or decrease over several cycles due to the
chemistry used. This produces a skewed noise level. Noise slope correction uses a line-of-best-fit to
determine the noise level instead of an average and normalizes to that line. Selecting this option
by clicking the
Slope Correct
button can improve data from replicates if sample baselines are
noticeably sloped. Noise slope correction improves the data when raw data backgrounds are
observed to slope upward or downward before the takeoff point (C
T
).
Where the slope is not steady or the initial cycles of the baseline show a significant increase or
decrease of the signal compared to the rest of the curve, Noise Slope Correction can lead to some
undesired effects, such as negative control curves crossing the threshold due to approximation of
the baseline as a line-of-best-fit and normalizing the raw data accordingly. As a consequence, this
function does not always improve the quality of the data and should be used only if the raw data
curves show a steady slope.
Takeoff point adjustment
The takeoff point adjustment algorithm can be used to define a minimum length of the baseline
utilized for normalization. In order to apply the takeoff point adjustment, two parameters have to
be defined. If a takeoff point is calculated by the
Dynamic Tube
that is lower than the first parameter,
then the second parameter is used as the takeoff point. The takeoff point adjustment can only be
used in conjunction with
Dynamic Tube
normalization.