Table 9: Potential problems, reasons and possible solutions
Step
Problem
Possible Reason
Preparing
completely
apoptotic
DNA.
Incomplete
conversion of
gDNA to
apoptotic DNA.
RF-10 has not been Warm RF-10 to 37°C.
pre-warmed to
37°C before 5 hr
incubation with
staurosporine.
Reactions
for
measuring
apoptotic
DNA.
Apoptotic DNA
reference curve
is not linear.
Solution
An apoptosis
inducer other than
staurosporine has
been used.
Use staurosporine, with which this
procedure has been optimised.
Staurosporine is a particularly
strong inducer.
Annealing/ligation
reactions were
prepared
incorrectly.
Due to the presence of
polyethylene glycol, reactions must
be mixed thoroughly to
homogeneity before annealing.
At the 10°C step after annealing,
the mixing of ligase into the
reaction is critical. Mixing is done
with the pipette tip, follows a
repeatable mixing sequence, and
without removing the tubes from
the Cycler. The tubes have to
remain cool to maintain linker
annealing and subsequent ligation.
Apoptotic DNA
standards have
been freezethawed several
times.
Freeze as single-use aliquots.
Apoptotic DNA
standards have
been stored frozen
at -20°C in a frostfree freezer.
Frost-free freezers will lead to
accumulation of condensate over
time, with concomitant changes in
DNA concentration. Freeze only at
-80°C and in a non-frost-free
freezer.
Table 9 continued
Step
Problem
Possible Reason
Solution
Reactions
for
measuring
apoptotic
DNA.
(continued)
Apoptotic DNA
reference curve
is not linear.
(continued)
Triplicate
threshold cycles
are not consistent.
Improve pipetting technique. Add
master mix into qPCR reaction
wells without employing the
pipette’s second stage expulsion
step (this eliminates bubbles while
still being reproducible, and is
faster). Then add diluted
annealing/ ligation reactions and
mix by aspiration reproducibly
from well to well. Use high quality,
calibrated pipettes.
Triplicate
threshold cycles
are not consistent
and baseline
fluorescence is
above 10,000
Synthesised linkers have been
supplied with dissolved
contaminants. Change linker
manufacturer.
PBMC do not
partition well
during Ficoll
gradient
centrifugation.
Blood contains
significant
amounts of lipid.
Change blood donor.
PBMC cell
counts are
inaccurate
and/or
inconsistent.
Washes of PBMC
have not been
sufficient to
remove platelets.
Wash 4 times to remove platelets.
Preparing
lysates for
the Cell
Number
standard
curve
Table 9. Troubleshooting strategies given here reflect problems that have occurred during
the development of this protocol.