Invitrogen Qubit 4 - Critical Assay Considerations

Qubit
™
4 Fluorometer User Guide 57
Critical Qubit
™
Assay considerations
How the Qubit
™
4
Fluorometer
calculates
concentration
The Qubit
™
4 Fluorometer generates concentration data based on the relationship
between the two standards used in calibration (three for the Qubit
™
protein
assay). The plot below shows the line corresponding to the curve-fitting
algorithm (a modified Hill plot) used in the calculation of concentration data for
the Qubit
™
RNA HS assay. For reference, the positions of the standards and a set
of data points from an actual experiment are shown superimposed onto the line.
This plot demonstrates that the curve-fitting algorithm gives accurate values for
quantitation.
Figure 1.
The curve-fitting algorithm used to determine concentration in the Qubit
™
RNA HS
assay. Data for other Qubit
™
quantitation assays are generated by similar algorithms.
Incubation time
To allow the Qubit
™
assay to reach maximum fluorescence, incubate the tubes for
the DNA and RNA assays for 2 minutes after mixing the sample or standard with
the working solution. After this incubation period, the fluorescence signal is stable
for 3 hours at room temperature for all nucleic acid assays except the Qubit
™
ssDNA assay, which is stable for up to 30 minutes.
The Qubit
™
protein assay requires 15 minutes of incubation for a stable signal. For
greatest accuracy in the protein assay, the incubation time of the samples should
be within 10 minutes of the incubation time of the standards.
Photobleaching of
Qubit
™
reagents
The Qubit
™
DNA and protein exhibit high photostability in the Qubit
™
4
Fluorometer, showing <0.3% drop in fluorescence after 9 readings and <2.5%
drop in fluorescence after 40 readings. It is important to remember, however, that
if the assay tube remains in the Qubit
™
4 Fluorometer for multiple readings, a
temporary reduction in fluorescence will be observed as the solution increases in
temperature (see Figure 2 in “
Assay temperature”, page 58). The RNA assays
including the RNA IQ assay should only be read once.
Note that the temperature inside the Qubit
™
4 Fluorometer may be as much as
3°C above room temperature after 1 hour. For this reason, if you want to perform
multiple readings of a single tube, remove the tube from the instrument and let it
equilibrate to room temperature for 30 seconds before taking another reading.