Top Ten Pitfalls in Quantitative Real-time PCR Primer Probe Design and Use

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 Applied Biosystems™ 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 Applied Biosystems™ TAMRA™ quenched probe and MGB-NFQ quenched probe are not completely interchangeable. (See # 4).

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.

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.

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.

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).

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.

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.

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.

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.

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.

REFERENCES

1. Reconstituting and Diluting Primers and TaqMan Probes (PDF)
2. Bioinformatic Evaluation of a Sequence for Custom TaqMan Gene Expression Assays (PDF)
3. Wootton JC, Federhen S (1996) Analysis of compositionally biased regions in sequence databases, Methods Enzymol 266:554–571.

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