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Top Ten Pitfalls in Quantitative Real-time PCR
Primer/Probe Design and Use
1. Primer and/or probe melting temperature (Tm)
is not optimal for the real-time PCR reaction.
The primer melting temperature (Tm) of each
PCR primer should be between 58–60°C, and TaqMan® probe Tm
should be ~10°C higher than the primer Tm. Also, the Tm of both
primers should be within 1°C. It is important to note that the
minor groove binder (MGB) moiety increases the Tm of the probe
by several degrees, so the sequence of a TAMRA™ quenched probe
and MGB-NFQ quenched probe are not completely interchangeable.
(See # 4).
2. Concentration of primers and/or probe are incorrect.
Reconstitute primers and probe into working
stock concentrations accurately. Ensure proper concentration
of the resuspended primer/probe by measuring the spectrophotometeric
absorbance at 260 nm. It is also important to take into account
the volumes that will be routinely pipetted (we recommend a minimum
of ≥5 µL) from working stocks of primers and probe when
setting up real-time PCR assays. A common range of working stock
concentrations for primers is 10–100 µM and for probes
is 2–10 µM. Accurate conversion of moles (mass)
primers to molar for reconstitution is necessary [1]. Example:
10,000 pmol + 100 µL H20 = 100 µM stock solution.
3. Primers and probes were designed against low
complexity sequence.
Regions of low-complexity sequence can create
problems in designing unique primer and probe sequences [2,3].
The best option would be to select an alternative region. If
that is not possible, choose longer primer and probe sequences
with higher Tm, to increase specificity. Also, optimization of
the thermal cycling protocol may be necessary to help reduce
nonspecific binding.
4. The probe was designed with MGB and ordered
as TAMRA, or vice-versa.
It is necessary to verify that the correct
probe (sequence, reporter, and quencher) is being used in the
real-time PCR assay. If the wrong probe is used, it is possible
that the Tm of that probe is incorrect for the real-time experiment.
This will greatly affect PCR efficiency and there is very little
thermal cycling optimization that may rescue the reaction.
5. Primers or probes were designed against low
integrity sequence.
Sometimes there are template sequence discrepancies/inaccuracies,
which can lead to failed assays caused by poor binding, or no
binding of primers and probes. It is important to verify the
sequence and check for the presence of single nucleotide polymorphism
(SNP) sites. It is recommended that multiple sequencing reactions
be performed to remove any sequence ambiguities, and to use public
databases with curated sequences such as NCBI (National Center
for Biotechnology Information) and dbSNP (Single Nucleotide Polymorphism
database) to determine the quality of the sequence [3]. Increasing
the primer length without increasing the annealing temperature
would allow for more wobble (primer-template mismatch).
6. Amplicon was too long.
Designing primers that generate a very long
amplicon may lead to poor amplification efficiency. Ideally,
amplicon length should be 50 to 150 bases for optimal PCR efficiency.
In cases in which longer amplicons are necessary, optimization
of the thermal cycling protocol and reaction components may be
necessary.
7. Primers and/or probes are designed against
wrong region of the gene.
Care should be taken to design the primers
and probes to the right target sequence, especially in the case
of known splice variants, mutant genes, or targets from a large
gene family, etc. Sometimes the primers/probe may span a wrong
splice site or perhaps not interrogate the correct transcript
from the gene family. If mutations are the target, probes should
ideally be designed with the mutation in the middle of its sequence.
Primer or amplicon sequences may be BLASTed (Basic Local Alignment
Search Tool) against the public database, to ensure that the
correct target is being amplified.
8. Primers and/or probes are designed against
wrong species.
If you are designing primers and probes to
targets from mixed source samples (e.g., transgenic samples,
pools of bacteria or viruses etc.), before starting the search
for primers and probes, check your target sequence for possible
homologies with sequences from other organisms by applying a
BLAST (Basic Local Alignment Search Tool) search. Public BLAST
servers are available on the internet (e.g. www.ncbi.nlm.nih.gov/BLAST).
The BLAST programs compare a query sequence to all sequences
in a specified database. To find specific primers and probes,
you should use only those target regions with minimum similarities
to other sequences
9. Target detection is not transcript-specific
and also amplifies background genomic DNA.
Genomic DNA (gDNA) is often co-extracted with
RNA and can therefore serve as a template in downstream processes,
such as PCR. False-positive results are obtained through amplification
of contaminating gDNA. Hence, it is preferable to have primers/probes
span exon-exon junctions (intron splice-sites) in the target
mRNA to prevent amplification of the target from contaminating
gDNA. In case of non-intronic sequences (e.g., from bacteria,
viruses, certain plant and mitochondrial sequences), wherein
such design criteria cannot be applied, it may be prudent to
use good RNA isolation techniques to minimize background gDNA
load and to treat the RNA sample with DNase before the reverse
transcription step.
10. Ordering a probe labeled with a dye not calibrated
or supported on the real-time PCR instrument being used.
Confirm that the probe was labeled with the
appropriate dye and verify that the dye used is calibrated and
supported on the real-time PCR instrument. A calibration may
be necessary before the dye can be used on the instrument.
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