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October 29, 2021 | PCR Solutions

Top 10 FAQs on optimizing dPCR assays on the QIAcuity

Webinar recap: QIAcuity Digital PCR Assay Optimization: Best Practices and Guidance

Our future is increasingly digital, and so is the way we look at science and healthcare. Forward-thinking laboratories are looking to adopt the latest technologies – many of which are based on digital answers. So, how can we make a valuable addition to the technological arsenal available in your laboratories today by introducing a rapid, scalable and straightforward digital PCR (dPCR) without rocking the boat too much?

You might be migrating your existing home-brew or commercial qPCR and dPCR assays or just getting started in dPCR. Like many bioanalytical systems, this requires optimizing assay conditions to ensure the highest level of performance. Led by our own John Chuckalovcak, Business Development Manager for digital PCR, the September webinar provided some best practices and tips on how you can do that on the nanoplate-based QIAcuity system with ease.

What your peers wanted to know…

To give you a glimpse, here are some of the frequently asked questions during the live Q&A session. If you couldn’t attend but would like to view the recording, you can sign up to watch it here.

What types of controls should I include in my pilot test for the assay?

We recommend using a well-characterized positive control for testing initial assay performance. Ideally, this control template would be present in the same background matrix as your actual samples. A No Template Control (NTC) is as critical as a positive control because it helps us detect any assay-specific artifacts or process-level contamination.

Do I need to run a full thermal gradient to optimize the annealing temperature of my assay?

No, a full thermal gradient is usually not required for optimization. Once you see two populations from your positive control sample dilution, running two additional tests at 2.5 degrees Celsius above and below that temperature is usually more than sufficient to resolve the intermediate positive partitions. This can be done quickly on the QIAcuity because each run takes only two hours to complete, with the four- and eight-plate systems producing data even faster for subsequent runs.

Is it necessary to adjust the annealing/elongation temperatures of established qPCR/dPCR assays when they are transferred to QIAcuity?

In most cases, well-established qPCR assays will work properly with the QIAcuity mixes (Probe and EvaGreen) without further adjusting the temperatures. We strongly recommend adhering to the cycling times as recommended in the corresponding manuals. For example, a qPCR assay might have been established using a master mix that contains a hot-start enzyme needing a long reactivation time at elevated temperatures, e.g., 10 min at 95°C. Such long exposure to high temperatures will be detrimental to the hot-start polymerase contained in the QIAcuity mixes.

Why do I see positive partitions in my no template controls? Is this contamination or something else?

The way to determine if you have contamination or assay artifacts is to look at the mean relative fluorescence unit (RFU) of the positive control samples. If you routinely see positive partitions in your NTCs that have the same fluorescence as your true samples, it is likely contamination. If the positives are of much lower amplitude, more random or diffused, it is likely an assay artifact. As always, our Technical Service and highly skilled Field Application Scientist teams are available to assist in your optimization.

What is the recommended dilution range of templates when testing new assays on the QIAcuity?

The ideal range is matched with your choice of Nanoplate partition density, either 8.5K or 26K. We can span from 17 to 170,000 copies per reaction and 6 to 220,000 copies per reaction for the 8.5K and 26K Nanoplates, respectively. We can use the positive control manufacturer’s specified concentration value for the gDNA template to guide our dilutions into this dynamic range. If using an in-house generated control, we can back-calculate the number of haploid or diploid copies present based upon the mass of the organism’s genome. For RNA or cDNA templates, the ideal range varies with the expression of the target of interest. A good rule of thumb is that for samples at Cq values of 25 or earlier, a 1:100 or 1:1000 dilution is a good starting point.

For RNA, is there a difference in selecting RT enzymes with and without RNase H (e.g., SuperScript vs. First Strand kit)? What is recommended?

We have had a good experience with both types of Reverse Transcriptases, e.g., the OmniScript RT, an RNAse H+ RT, which is part of the QuantiTect Reverse Transcription Kit, as well as with RNase H- enzymes such as the various SuperScript enzymes.

Where is the best place to set my threshold to differentiate positive and negative partition populations?

The position of your threshold should be informed by the performance of the No Template Control (NTC) reactions. Through running several NTCs, we define the mean RFU of the negative population and the variation around it. Thresholds should be set high enough above the negative population to avoid routinely including negative partitions in the positive partition space.

What is the best dye or channel to use on the QIAcuity?

The QIAcuity has five active channels to use for detection. QIAGEN has validated the system to ensure optimal performance across all these channels. Therefore, you can feel confident using any of the dyes recommended in the QIAcuity User Manual Extension: QIAcuity Application Guide.

Is dye calibration included for all channels? And do you provide plates or solutions for this purpose?

Calibration is an integral part of the software, given that the recommended dyes are used. No dedicated calibration plates or solutions are required.

Do you recommend EvaGreen or hydrolysis probe detection?

Both chemistries have their place in dPCR, and the choice depends on your applications and needs. Common considerations are multiplexing requirements, target homology and cost.

Hydrolysis probes allow the detection of multiple targets per well, which is beneficial with limited samples or when the discrimination of highly homologous sequences is required (e.g., SNPs.) These benefits are weighed against the cost of an additional, fluorescently labeled oligo per target and the more stringent design requirements.

Primers and EvaGreen dye are well-suited for applications that do not require multiplexing or justify the investment in a probe. Two examples are gene expression and copy number variation analyses, wherein target and reference genes can be run in separate wells.

Check out our beginner’s guide to learn more about how QIAGEN can support your transition from qPCR to dPCR.

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