Biomedical Engineering Reference
In-Depth Information
—Consider if there is too little cDNA template in the qPCR. For a medium to highly
expressed gene, use the equivalent of 0.5
μ
l of first-strand cDNA synthesis reaction).
If a very late
C
q
is recorded, add up to 2.5
μ
l of cDNA synthesis reaction in a 25
μ
l
qPCR.
—Consider if there is there too much cDNA template in the qPCR. If the final con-
centration of the cDNA synthesis reaction components in the PCR is too high this is
inhibitory to PCR. Therefore, limit the volume of the cDNA synthesis reaction added
to 10% of the volume of the PCR.
—Check to see if the RNA is degraded. Use control cDNA to confirm that the assay
works and an alternative qPCR assay to confirm that the samples support RT-qPCR.
—Consider the length of the amplicon. Ideally this should be
<
120 bp, especially for
FFPE tissue samples. Longer amplicons amplify with lower efficiency and should be
avoided.
—During RT, after denaturation of RNA, ensure that the RNA is kept on ice.
—If using homemade buffers, ensure that the salt and buffer concentrations are correct.
—Incomplete thawing of frozen 2
master mix buffers will change the salt concentration
in the remaining buffer. Increases in MgCl
2
concentrations will affect the efficiency
of primer binding and may cause the appearance of primer dimers and reduce the
efficiency of the PCR. Ensure that master mixes and stock reaction components (oligos,
MgCl
2
) are completely defrosted and thoroughly mixed before use.
—If there have been changes in the reaction volumes or number of reactions for which
master mix has been prepared, then errors may have been made during pipetting.
Changing the volumes of the reaction mixtures can cause different amounts of error
in the volumes being dispensed. This is partially due to differences in the tolerances
between large- and small-volume pipettes. This error may not be propagated linearly
during scale up. Highly sensitive methods such as PCR can significantly magnify these
problems. It is best to scale up in stages if absolutely required. A pipetting robot can
be a wonderful addition to the routine laboratory and these are particularly useful for
high-throughput applications and to avoid variability in reaction mixes.
×
•
Check the reagents and thermocycler.
—If the assay is probe based and has previously worked well, then the probe may have
degraded. For example, it may have been photo bleached if it has been left in the
light. In order to check a probe function, digest an aliquot (alongside a non-digested
control) using DNAse I (use extreme care when handling DNAse I in close proximity
to oligonucleotides). Examine the fluorescent yield and compare with a functioning
assay. If the fluorescence is significantly lower then a new probe must be used. To
prevent degradation, always store fluorophore-labeled oligonucleotides in aliquots in
the dark at
20
◦
C. These can be used for at least 6 months after resuspension.
−
—If the background fluorescence level of the probe is very high, then it is possible that
it may have become hydrolysed; for example, if subjected to repeated freeze-thaw
cycles. See above for a diagnostic test.
