Frequently asked
questions

Our unique technology, Crystal Digital PCR™, relies on sample partitioning into 2D monolayer arrays of droplets, which are assembled into each of the rectangular chambers of the Sapphire chip. Within this chamber, we can observe that the droplets self-order into a periodic arrangement reminiscent of the pattern found for atoms within crystalline structures. This is why we call the droplets within the chamber droplet crystals.

You may refer to our naica® system user manual (can be found here) for guidelines, a whole host of descriptive items, and step-by-step tutorials on how to set up digital PCR experiments on gene-pi.com, an educational website equipped with resources and information about digital PCR. We would also like to refer you to the digital MIQE guidelines  (Huggett et al, 2013) for additional considerations when setting up and reporting results from digital PCR experiments.

In real-time PCR, quantification is achieved using a standard curve or is expressed relatively to a reference sample. In digital PCR, you can obtain absolute quantification without the need for references. Moreover, when attempting to detect or quantify rare events in a complex sample, such as a rare mutant in a background of wild-type DNA, amplification bias may occur due to competition for reagents, and result in the impossibility to detect this rare mutant when using traditional real-time PCR. By segregating target molecules in different compartments, digital PCR alleviates or eliminates competition and amplification bias, thus allowing detection of even a small fraction of rare targets. Finally, it has also been shown that digital PCR is more tolerant of some inhibitors commonly found in clinical or environmental samples, ensuring accurate quantification even in samples where either complete or partial inhibition had been observed in real-time PCR. Check out our  Application Note comparing the precision performance of Crystal Digital PCR to qPCR here.

In real-time PCR, fluorescence intensity is measured at each cycle, and is compared to the fluorescence pattern measured for standards or references for quantification. This is thus an analog measurement. Digital PCR on the other hand will mesure discrete values, the number of positive partitions and the number of total partitions, leading to absolute quantification of the target.

Digital PCR is a novel implementation of PCR that relies on dividing the initial reaction mix into multitude of smaller reactions. In Crystal Digital PCR, this is achieved through dividing the reaction mix into tens of thousands of subnanoliter droplets. DNA molecules thus partitioned into droplets are amplified separately, and a fluorescent reporter, such as a hydrolysis probe, allows enumeration of the droplets in which amplification and probe hybridization has taken place. The fraction of droplets thus deemed positive for amplification is recorded, as is the total number of fractions analyzed (total number of droplets). Using Poisson’s law of small numbers, the concentration of target DNA can then be approximated, with the final results given as a mean concentration of copies of targets/ unit volume, and the uncertainty associated with this specific measurement. You can read in more detail about the principle of digital PCR here and watch this video with a detailed illustration of the digital PCR process.

Yes, you may image the chips prior to amplification, please refer to the “Imaging Droplets Crystal Prior to PCR” protocol for instructions available here: https://www.stillatechnologies.com/technical-resources/(secured access) or contact the support team at support@stilla.fr or support@stilla-inc.com (North America).

Yes, you can recover droplets after PCR. To request the protocol, please contact support@stilla.fr or support@stilla-inc.com (North America). You can also read the Application Note on “Droplet Recovery” here https://www.stillatechnologies.com/droplet-recovery-with-crystal-digital-tm-pcr/

If you previously ran this assay on a qPCR, you may try using the same hybridization temperature. If separability between negative and positive populations is not adequate (presence of rain, populations are close to one another), you may then optimize the assay by testing different hybridization temperatures. Do not hesitate to contact the Support Team if you need additional help at support@stilla.fr or support@stilla-inc.com (North America), we’ll be glad to assist you.