Digital PCR Support—Getting Started

The QuantStudio® 3D Digital PCR System is the ideal system to perform digital PCR. The QuantStudio® 12K Flex System with OpenArray® block is also designed for digital PCR applications.

Digital PCR can be used to perform rare allele detection, low-fold gene expression determination, viral titering, absolute quantitation of next-generation sequencing libraries, pathogen detection, generation of reference standards, GMO monitoring, and copy number variation. While we don’t have step-by-step workflows for all possible applications, we are working on rolling these out over time.

The Poisson distribution, when applied to digital PCR, describes the relationship between the percent of negative reactions and the average copies per reaction. This phenomenon was first described by Simeon Denis Poisson.

The digital range is the template dilution range in which your results are a mix of both positive and negative wells (i.e., the green area of the curve below). It is important for your sample to be within the digital range for accurate quantitation. The most accurate region occurs when you have on average one copy per reaction, which equates to ~37% negative reactions.

 Please see the section on Sample Dilution (p. 24) in the QuantStudio® 3D User Guide for more details on how determine the target copy number per genome.

For the QuantStudio® 3D Digital PCR System we recommend the QuantStudio® 3D Digital PCR Master Mix (Cat. No. 4482710).
We recommend the TaqMan® OpenArray® Digital Master Mix for the QuantStudio® 12K Flex OpenArray® System. This master mix is available in the TaqMan® OpenArray® Digital PCR Kit (Cat. No. 4460585).

Using the QuantStudio® 3D Digital PCR System, each chip will contain one sample. Up to 24 chips can be run at one time on the thermal cycler, and to read each chip takes about 30 seconds. The whole processing of the chip from the read takes about 1 minute.

When using the QuantStudio® 12K Flex OpenArray® System, you have the flexibility to run multiple samples per OpenArray® plate (such as 48, 24, 16, 12, or 1 sample per OpenArray® plate). The exact number of samples to run per OpenArray® plate would be determined by the number of replicates needed and the level of confidence you desire from your results.

The QuantStudio® 3D Digital PCR System is a small, economical instrument designed specifically for digital PCR. It uses a small silicon-based chip with 20,000 reaction wells—one sample per chip. The QuantStudio® 3D Digital PCR System reads the endpoint fluorescence from the digital chip—cycling is performed offline on a compatible thermal cycler. Analysis is performed using the AnalysisSuite™ Software.

The QuantStudio® 12K Flex OpenArray® System is a real-time PCR instrument that reads an OpenArray® plate with 3,072 reaction through-holes. It can be used for various applications and with different block types, including an OpenArray® block for use in digital PCR. Both cycling and the read are performed on the QuantStudio® 12K Flex System. Up to 4 OpenArray® plates can be run at one time, which each plate containing either one or multiple samples. Analysis is performed using the DigitalSuite™ Software.

The QuantStudio® 3D Digital PCR instrument is capable of detecting dyes that emit at 520 ± 10 nm (e.g., FAM™, intercalating dyes such as SYBR® dye), 558 ± 7 nm (e.g., VIC®), and 630 ± 30 nm (e.g., ROX™ dye, which is used as an internal reference dye). Because digital PCR is an endpoint solution and does not measure signal in real time, it is important to have confidence in endpoint readouts, and thus, our protocols utilize TaqMan® assays to ensure amplicon specificity. Intercalating dyes can be used; however, it is important to have a method of verification. We recommend and support TaqMan® probe–based methods, which are much more specific. For laboratories that are budget-conscious or have already established SYBR® primers, a working R&D protocol is available.

Please see our comparison table here of the different PCR technologies.

Yes, the current Chips will work with the new v2 Chip Lids.

Yes, the current Lids will work on the new v2 Chips.

No, the current v1 Master Mix can only be used with the older Chips.

No, the v2 Master Mix can only be used with the v2 Chips.

We recommend you upgrade the firmware to v3.0 in order to read the 2D barcode; otherwise the .eds files will not have a Chip ID.

No. No matter which version of Chip Lid is being used, v2 Chips require Firmware v3.0 for proper data analysis.

Yes, Firmware v3.0 will work with all versions of Chips and Lids.

Yes, internal studies have shown comparable performance between v1 and v2 Chips.

No significant difference has been measured.

At low concentrations, there is no impact on the data. At higher concentrations (>2000 copies/L), there is a slight impact. Using the Poisson Plus algorithm will correct for well-to-well volume variation.

Yes, please use new Cat. No. A27322.

You should use the Poisson Plus algorithm for projects that contain QuantStudio™ 3D Chips with target quantities >2000 copies/μL.

The Poisson Plus algorithm corrects for well-to-well load volume variation, on a per Chip basis. This becomes important at higher target concentrations.

Yes, there is an option to export the Chip data as XML on the Export tab in AnalysisSuite™ software v3.0.