Biomedical Engineering Reference
In-Depth Information
•
sequence and position of primers and probe (if appropriate);
•
precise details of the RT reaction, especially the amount of RNA and priming strategies
used;
•
amount of RT reaction transferred to qPCR reaction;
•
qPCR conditions;
•
efficiency and lowest limit of linearity for the assay (molecules and
C
q
);
•
error at that limit;
•
normalization procedure and justification for reference genes selection;
•
when reporting relative quantification data, the range of
C
q
values covered should be
included.
6.3 Troubleshooting
6.3.1 No/Poor/Late amplification
No amplification is characterized by no significant increase in fluorescence above back-
ground. Poor amplification is typified by very low relative fluorescence values (
R
n
;
<
0.05)
with either the control templates or samples, or by a slope that varies significantly between
samples amplifying the same target. A sample generating a
C
q
>
35 should be regarded
with care, since this is very late and indicative of a theoretical, initial concentration of
approximately 10 copies.
•
Check the qPCR reaction conditions.
—Check the amplification product on a gel. If no product is present, then repeat the
assay, checking that all reagents are added and that the thermal cycling conditions are
correct. If a product is present, then the instrument detection settings may be incorrect;
for example, the wrong filter may be used to detect the light emitted from the reporter
fluorophore. Collect all emitted data using all available data-collecting channels or
filters. Check detection by substituting a probe with SYBR Green I dye to determine
if the primers are amplifying.
—Ensure that all required reagents (i.e. enzyme(s), reaction buffer, primers, probe and
template) were added.
—Check the annealing temperature (
<
60
◦
C), elongation times and cycle number. Ensure
that the correct activation time for the DNA polymerase is used (antibody-inactivated
enzymes usually require shorter initial incubation periods than chemical-inactivated
DNA polymerases)
—Check that the primers and other reactants have been diluted correctly.
—Ensure that the pipettes are calibrated.
—Ensure that the thermal cycler is programmed to detect fluorescence during the anneal-
ing and extension stages of the PCR.
