Biomedical Engineering Reference
In-Depth Information
—If the assay is a new assay, then it may well be that the probe manufacturer is to blame.
Test the quality of the probe using a DNAse I digestion assay and compare with the
fluorescence from a functioning probe (as above); following digestion the fluorescence
should greatly increased due to reporter and quencher becoming separated. When the
fluorescent yield is negligible, return the probe to the manufacturer and request a
replacement.
—SYBR Green I dye loses fluorescent emission after storage. After SYBR Green I
has been diluted, it goes off very quickly and can only be kept in the fridge for
approximately 2 weeks. It must also be kept in the dark. For best results, dilute a fresh
batch of SYBR Green I dye each day or use a commercial master mix, which usually
contains components that stabilize the fluorescence.
—If using a new batch of thermostable DNA polymerase (or 2
master mix buffer con-
taining DNA polymerase), be aware that different batches of DNA polymerase (quite
apart from DNA polymerases from different suppliers) can have different polymerase
and exonuclease activity, but still be within the manufacturer's specifications. Where
possible, use the same batch of reagents and plan experimentation such that target
quantification in all samples to be compared is carried out in as short a time period as
possible. Use positive controls and standard curves to compare batches of master mix
or enzymes.
×
—Different thermocyclers (particularly if they feature heating blocks) and thermocyclers
from different manufacturers have different ramping kinetics and heating efficiencies
and, hence, can affect the efficiency of the PCR. Variability in heating efficiency may
also occur in different wells, and particularly so in 96- or 384-well instruments. When
adapting a protocol, which has been optimized for a given instrument, ensure that
conditions are optimized for the instrument to be used.
•
Ensure the optimal design of RT primer, and qPCR primers (and probe), and optimal
reaction conditions.
—Ensure that the RT primer is not binding to a region of the RNA molecule that could
be double stranded. If this is the case, move the assay to an alternative region of the
gene and use an alternative gene-specific primer.
—When using gene-specific RT primers, make sure they specify the anti-sense sequence.
—When using oligodT RT priming, ensure that qPCR assays are directed to approxi-
mately 1 kb from the polyA tail or 3
end of the target sequence in order to ensure that
the cDNA contains the representative sequence.
—Denature/anneal RNA and primers in the absence of salts and buffer.
—Ensure that RT was performed at the right temperature (check manufacturer's recom-
mendations for each enzyme).
—Raise the RT reaction temperature to 65
◦
C if the RT primer
T
m
and the reverse
transcriptase allows.
—When performing a two-step RT-qPCR protocol, ensure that the volume of RT reaction
mixture added to the qPCR does not exceed 10% of the volume of the qPCR.
