14. Usually the standard curve for high concentrations is sufficient.
In case of doubt, prepare both standards.
15. Depending on the plate reader, the fluorescence intensity
should be adjusted to the highest standard available.
16. Usually, the standard error for a qPCR analysis makes mass
balances difficult, 80–120% are still in a normal range.
17. In some cases, sums exceed 100%. This can have different
reasons. (1) Check the interfering substances in the manual
of the DNA extraction as well as the qPCR kit. Some chemicals,
such as salts, may interfere with the assay, and result in higher
or lower apparent concentrations. (2) Check the linearity of
concentration-dependent qPCR measurements. Sometimes,
very low or very high concentrations can lead to a big relative
error.
18. In other cases, recovery rates are constantly low. In this case,
the reasons are due to very complex interactions. (1) By modifying viruses, as done in pseudotyping, size and surface properties may change as well, which leads to altered adsorptive
properties. An evaluation of membrane material and MWCO
should be executed carefully in static and dynamic experiments.
(2) Low recovery rates in the clarification process may indicate
that cell lysis is not optimal. Hence, virus particles still adhere
to cell debris, and are excluded in the filtration process.
(3) Shear forces may be too high and the virus is lost.
19. Depending on the regeneration of the virus, values should be
expressed in log-scale differences.
20. This value can indicate whether the point of harvesting and the
cell lysis are optimal. Some viruses need to bud to fully assemble and become infectious, whereas infectious baculoviruses are
formed in the cell and by budding. Furthermore, released
viruses, by budding or lysis, may decay in the cultivation
broth due to heat, UV radiation, or more complex processes.
21. If this ratio decreases due to an increased passaging, reasons
could be sought in the presence of defective interfering
particles.
22. If the reduction of dsDNA and total protein is not sufficient for
the desired application, different approaches can be performed.
Either a nuclease treatment, to reduce the size of DNA fragments and to facilitate DNA reduction, or a subsequent process
step such as a chromatographic method can be used.
23. Wash cells very gently, so as to not detach them.
24. Verify the detachment of the cells with an incident light microscope. If the cells are not round, and still stick to the surface,
increase the detachment time.
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Friederike Eilts et al.
In case of doubt, prepare both standards.
15. Depending on the plate reader, the fluorescence intensity
should be adjusted to the highest standard available.
16. Usually, the standard error for a qPCR analysis makes mass
balances difficult, 80–120% are still in a normal range.
17. In some cases, sums exceed 100%. This can have different
reasons. (1) Check the interfering substances in the manual
of the DNA extraction as well as the qPCR kit. Some chemicals,
such as salts, may interfere with the assay, and result in higher
or lower apparent concentrations. (2) Check the linearity of
concentration-dependent qPCR measurements. Sometimes,
very low or very high concentrations can lead to a big relative
error.
18. In other cases, recovery rates are constantly low. In this case,
the reasons are due to very complex interactions. (1) By modifying viruses, as done in pseudotyping, size and surface properties may change as well, which leads to altered adsorptive
properties. An evaluation of membrane material and MWCO
should be executed carefully in static and dynamic experiments.
(2) Low recovery rates in the clarification process may indicate
that cell lysis is not optimal. Hence, virus particles still adhere
to cell debris, and are excluded in the filtration process.
(3) Shear forces may be too high and the virus is lost.
19. Depending on the regeneration of the virus, values should be
expressed in log-scale differences.
20. This value can indicate whether the point of harvesting and the
cell lysis are optimal. Some viruses need to bud to fully assemble and become infectious, whereas infectious baculoviruses are
formed in the cell and by budding. Furthermore, released
viruses, by budding or lysis, may decay in the cultivation
broth due to heat, UV radiation, or more complex processes.
21. If this ratio decreases due to an increased passaging, reasons
could be sought in the presence of defective interfering
particles.
22. If the reduction of dsDNA and total protein is not sufficient for
the desired application, different approaches can be performed.
Either a nuclease treatment, to reduce the size of DNA fragments and to facilitate DNA reduction, or a subsequent process
step such as a chromatographic method can be used.
23. Wash cells very gently, so as to not detach them.
24. Verify the detachment of the cells with an incident light microscope. If the cells are not round, and still stick to the surface,
increase the detachment time.
384
Friederike Eilts et al.