Biomedical Engineering Reference
In-Depth Information
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Use fresh aliquots of all reagents, including sterile water.
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Only use pipettes, tips, solutions (especially water) and racks dedicated to setting up
qPCR. When possible, use a protective laminar flow hood. Do not use pipettes and
accessories that have been exposed to amplicons.
•
Decontaminate surfaces, pipettes and racks using UV exposure for 5-10min prior to
reaction set-up.
•
Change gloves (after a maximum of 30min wear) and tips frequently.
•
Change the location of PCR set-up.
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When using SYBR Green I detection, check the melt curves. If the non-template control
melt curve is different from the amplicon melt curve, you may still be able to use the
template-derived data.
•
An excess of probe can generate an artifactual positive result. This is often distinguished
from a genuine positive by examination of raw data and comparison of background
fluorescence. An artifact is apparent by a linear increase in background fluorescence that
does not have a logarithmic phase. Alternatively, where a result is in question, repeat the
assay on the sample using lower probe concentration (down to 50 nM).
6.3.3 No reverse transcriptase control yields an amplification product
A positive 'No RT control' indicates that the RNA sample contains contaminating DNA.
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To remove gDNA contamination, treat RNA samples with RNAse-free DNase I.
•
If possible, design PCR primers to amplify across introns to avoid detection of genomic
DNA template.
6.3.4 Primer dimers formed
Primer dimers are more likely to occur in no template controls or when there is very little
target RNA. Even small amounts of target can suppress primer dimer formation.
•
If dimers occur in the presence of normal amounts of template RNA, then redesign PCR
primers. Ensure that complementary sequences are absent from the 3
ends of the primers
(see Section 6.2.5 for details).
6.3.5 Multiple peaks in SYBR green I melt curve
•
Improve the stringency of the qPCR to avoid non-specific hybridization by raising the
annealing temperature or lowering the MgCl
2
concentration.
•
Treat the RNA with RNase-free DNAse I.
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Primers may be amplifying multiple genuine products (e.g. splice variants, pseudogenes)
and the assay may require primer redesign in order to detect the specific target. Analyze the
reaction products by gel electrophoresis to check for specificity and consistent presence
of additional bands. Perform BLASTn analysis to identify potential
in silico
products
when target sequences are fully described.
