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Tips for qPCR Assay Design
As with any PCR-based technology, assay design is crucial to obtaining high-quality qPCR data. First, place primers properly: robust amplification starts with well-designed primers for amplicons about 70–150 bp long that display no secondary structure, which can be verified using programs like MFOLD. If you assess gene expression in eukaryotic cells, design your primers to span an exon-exon junction to avoid amplifying any contaminating genomic DNA that may have carried over from DNA purification. Once you've selected your target, verify specificity with an online tool, such as Primer-BLAST, to confirm that it is unique.
For target amplification, ideal primers will have a GC content of 40–60% and a melting temperature (Tm) between 50–65°C, as predicted using a Tm calculator such as http://www.basic.northwestern.edu/biotools/oligocalc.html. For targeted amplification, avoid designing primers in which a single base repeats four or more times. In addition, check that sequences of the forward and reverse primers do not have 3' complementarity, as this can result in primer-dimer formation.
Probe design is an equally important component of a qPCR assay. Design probes with melting temperatures 8–10°C higher than those of the primers. Probes should be 30 nucleotides or shorter for most applications, but if the probe is longer, consider using an internal quencher. Ensure your probe does not have a G at its 5' end, which can quench fluorescence even after hydrolysis. If you are assessing gene expression in eukaryotic cells and have not designed either primer to span an exon-exon junction, then consider designing your probe to span the exon-exon junction instead. As with your primers, run the probe sequence through a Primer-BLAST alignment to ensure the sequence is unique and will amplify only your target sequence of interest.
Tips for ddPCR Assay Design
Best practices for designing ddPCR primers are similar to but distinct from those of qPCR. Primers should have a GC content of 50–60% percent with a Tm between 50–65°C. Avoid repeats of Gs or Cs longer than three bases, and try to place Gs and Cs at the 3' nucleotide of primers whenever possible. As with the qPCR assay, avoid secondary structure in primers and the target sequence and 3' complementarity of primers to prevent primer-dimers. Programs like Primer3 (Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology) simplify the process of selecting optimal primer sequences.
Our ddPCR systems support hydrolysis probe (TaqMan®) and DNA binding dye (EvaGreen®) assays, which include a sequence-specific, fluorescently labeled oligonucleotide probe in addition to the sequence-specific primers. Proximity to the reporter quenches the intact probe, but the amplification reaction separates the reporter from the quencher, producing a fluorescence signal proportional to the amplified product in the sample.
When designing probes, choose a sequence between the two primers of the amplicon. Primer sequences cannot overlap with the probe, though they can sit directly next to one another. The Tm of a hydrolysis probe should be 3–10°C higher than that of the primers and the GC content should be 30–80%, but keep in mind that the probe should have more Cs than Gs and that the probe should not have a G at the 5' end because this quenches the fluorescence signal even after hydrolysis. The distance between the fluorophore and quencher affects baseline signal intensity, so the probe should be between 13 and 30 nucleotides long. Shorter probes discriminate better between single base differences in the target amplicon(s), but these may have a lower Tm, so we recommend longer probes or Tm enhancers if needed to achieve a high enough Tm. Furthermore, we recommend using Tm enhancers for SNP probes and rare mutation detection assays to keep the background fluorescence minimal.
Best Practices for Both Technologies
Once you've designed a qPCR or ddPCR assay, following laboratory best practices can maximize the likelihood of successful, reproducible reactions and avoid common pitfalls. Avoid contamination by cleaning your bench with 10% bleach—not ethanol. Ethanol only precipitates DNA and spreads it around on surfaces. When setting up an assay, wear gloves, work in a dedicated PCR area, and use dedicated pipets for qPCR and ddPCR. When it comes to reagents, always use PCR-grade water and aliquot all components of the PCR reaction for single-time use. Lastly, we recommend using screwcap tubes for templates and aerosol-resistant filter tips to prevent contamination from aerosolized nucleic acids.
To improve reproducibility, keep your pipets calibrated. Avoid pipetting less than 5 microliters; prepare enough master mix to run all your reactions plus 10% extra. Do not freeze and reuse. The master mix should contain all reaction components except the template: mix thoroughly and dispense into each well. This way, reactions occur in an identical chemical environment.
Your reaction will be only as good as the template DNA or RNA you start with. Before use, assess the quality of your nucleic acid—for example, by heating it to above 60°C, using gel electrophoresis, or bioanalyzer analysis. Using a degraded or contaminated template for a qPCR reaction will produce poor reaction efficiencies and low-quality data that are analytically inaccurate. And while ddPCR reactions are less susceptible to PCR inhibitors, carefully removing them when preparing your nucleic acid samples is still a good idea.
Finally, you need controls to gauge the specificity and accuracy of your assay or troubleshoot if something goes wrong. Therefore, include a positive control that contains a verified target template, a control without the template, and a negative control without enzyme—as well as any other controls appropriate for your specific assay.
When deciding between qPCR and ddPCR to measure gene expression, only you know which best suits your experimental needs. While similar in some ways, these technologies have different strengths and limitations that make a choice highly situational. Once decided, you can follow a well-defined path forward for either assay type. By following best practices, you can use these assays to reveal the insight necessary to help your research reach new heights.
BIO-RAD, DDPCR, and DROPLET DIGITAL are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. TaqMan® probes are dual labeled hydrolysis probes and are a registered trademark of the Roche Molecular Systems, Inc. EvaGreen is a trademark of Biotium, Inc. All trademarks used herein are the property of their respective owner. © 2023
