Frequently asked
questions
When performing a Crystal Digital PCR experiment, you only need the Sapphire chips and PCR caps. Everything you need is conveniently contained within the Sapphire chips box delivered to you.
We do not recommend such usage, mostly due to concerns over potential contamination. Indeed, we do not advise that any consumable that has been in a post-PCR environnment is returned to a prePCR lab.
After your Crystal Digital PCR experiment, you may conserve your Sapphire chips for up to a week.
Yes, you may perform a run overnight and read it the next day. This will not affect your results.
Sapphire chips are stored at room temperature (19-25°C).
Based on our results obtained with the PerfeCTa mix, the droplet size variation is 2.8% (droplet diameter) or 0.44 nL +/- 0.03 nL.
The Naica system is For Research Use Only. However, depending on the legislation of your country, you may be able to use the Naica system for diagnostics purposes. Please check with your national Health Authority whether this is the case.
No, the Naica system is not currently marked CE-IVD, but we are working on it!
Yes, the Geode and the Prism3 are both CE-marked.
The Naica system is a versatile system, which you can use to perform digital PCR, one-step RT-PCR, Whole Genome Amplification. You can use as starting material DNA, RNA, cells or … anything else! Please contact us at support@stilla.fr so we may help you set up experiments. Lastly, using our Sapphire chips, you can recover droplets after you have imaged the chips, should you need the amplified material for downstream analysis. You can also image the chambers prior to amplification, using either our Prism3, or any other means of imaging such as an inverted fluorescence microscope.
You may analyze up to 12 samples per run per Geode using our Sapphire chips.
The Prism3 has three detection channels, which can be used to reliably quantify three different targets. However, you can increase the multiplexing capacity by grouping targets and detect them using one detection channel, if your application allows such detection strategy.
If equipped with a single Geode and a Prism3, you may perform up to 4 runs per day, meaning you can analyze up to 48 samples. Since the combined partitioning/ thermocycling step performed by the Geode is the bottleneck, you can purchase additional Geodes to increase throughput, doubling the number of samples analyzed per day for each Geode installed.
Please make sure the Geode hot plate remains free of dust, by cleaning it using a keyboard air spray. For more information on maintenance contracts, please contact our commercial team at sales@stilla.fr
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 for guidelines on how to set up digital PCR experiments. 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.
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.
No. Stilla only provides the instruments, the chips, the analysis software… and the expertise! Please contact your local distributor for any of the PCR reagents needed for Crystal Digital PCR.
You may purchase a Naica System, additional instruments or Sapphire chips through sales@stilla.fr.
You may purchase a Naica System, additional instruments or Sapphire chips through sales@stilla.fr.
You may purchase extra PCR caps for Sapphire chips through order@stilla.fr.
You may purchase Sapphire chips through order@stilla.fr.
At this time, we only offer Sapphire chips. Please note that we are currently developping an alternative consumable which allows the analysis of up to 48 samples / run.
Using the Sapphire chips, you may quantify from 0.2 copies/ uL to 20,000 copies/ uL.
Block uniformity of the Geode is +/- 0.5°C at 72°C.
The Geode can be operated from +4°C to 95°C.
The total number of droplets analyzed is between 25,000-30,000 per chamber. More droplets are generated, but are excluded on QC criteria.
The droplets generated within our Sapphire chips are very stable, and remain intact for up to a week after the experiment.
The droplets generated have a diameter of 94 microns, which equates to a mean volume of 0.44 nL.
Please make sure the Prism3 tray and chip holder remain free of dust, by cleaning them using a soft, lint-free tissue. For more information on maintenance contracts, please contact our commercial team at sales@stilla.fr
The Prism3 has 3 detection channels: Blue (Ex 415-480 nm; Em 495-520 nm), Green (Ex 530-550 nm; Em 560-610 nm), Red (Ex 615-645 nm; Em 655-720 nm).
The Naica Prism3 is a compact instrument with dimensions of just 44 x 34 x 21 cm or 17 x13 x8 inch (W x D x H), weight: 15 kgs.
Yes, Stilla is able to provide you with an appropriate compressed air system, depending on the number of Geodes that need to be connected to the compressed air system.
The Naica Geode is a compact thermocycler with the following dimensions: 35 x 37 x29 cm or 14 x 15 x 11 inch (W x D x H), weight: 18 kgs.
The throughput for a standard Naica system (1 Geode and 1 Prism3) is 12 samples every 2h30, or 48 samples per day. Please note that you can easily increase your throughput by adding one or more Geode to this set-up.
The Naica system is composed of a Geode (35 x 37 x29 cm or 14 x 15 x 11 inch), a Prism3 (44 x 34 x 21 cm or 17 x13 x8 inch), a standard PC and a pump (25 x 25 x 35 cm or 9.8 x 9.8 x 14 inch).
You may use any fluorophore you wish with the Naica system, provided that their spectra fall within the excitation/ emission spectra of the Prism3. Blue channel: Em 415-480 nm/ Ex 495-520 nm ; Green: Ex 530-550 nm/ Em 560-610 nm; Red: Ex 615-645 nm/ Em 655-720 nm.
Fluorescence spillover describes the process by which a fluorescent signal, aimed to be detected only in a given detection channel, is also detected in the adjacent detection channel. This can lead to errors in quantification, it is therefore extremely important to correct this post- acquisition by applying fluorescence spillover compensation. Please refer to the Crystal Miner user manual for detailed instructions on compensation.
QC Flags are evaluated on three criteria: Number of analyzable droplets; Image Sharpness (whether the image is in focus or not); Number of Saturated Objects (whether some objects have a fluorescence of >65 536 RFU). There are three types of flags : Green: high quality; Green/ Yellow: moderate quality; and Yellow: Low quality. Please note that the QC flag for each sample will always take the worst individual flag status. Similarly for the experiment: if one of the chambers has got a yellow flag, the experiment as a whole is automatically assigned a yellow flag.
Visualize the droplets crystal of your chip by using the mouse scroll to zoom in the image : it should not be blurred. The flag corresponding to the sharpness should be green. If it is green and orange or orange, here is how to proceed :
- please verify that the chip or/and the holder are clean. If not, use a tissue and re-scan.
- please verify that the chip is on a flat position
If you hesitate, please send us the picture so we can check on the image sharpness.
We recommend that you change the focus by doing an automated focus calibration : place a chip (containing droplets with at least fluorescein in the B or C chamber) in the second position of the holder.
Then log in as a manager, go in the “Settings” window and click on the “focus calibration” button. Select the chamber that will be scanned for focus calibration (B or C in the second position of the holder) and validate. A progress bar will be displayed with the estimated remaining time. At the end of this procedure, the software will suggest the best focus value. Please refer to pages 28-29 in the Crystal Reader user manual.
No, the focus will be set at installation of your Naica system. You do not need to set it again, unless the Prism3 has been exposed to shocks.
It is expressed in mm and it corresponds to the depth of the imaging plane used for image acquisition.
The automated estimation is performed such that you obtain the maximum separability between the positive and the negative droplet populations, while minimizing the intra-population value dispersion. You can manually adjust it (by right-cliking on the threshold) but please note that within an experiment, the threshold value will be the same for all chambers.
To install the Crystal Miner software on your PC, plug the Stilla USB key and go on the “CrystalMiner Installer” directory : install first the prerequisites located in the “prerequisites” folder, then install the Crystal Miner application.
Operating system : Windows 10 in 64 bits (recommended) or Windows 7 in 64bits. RAM : at leat 16Go. Processors : at least 2cores of 2GHz or higher. Screen resolution : at least 1920×1080; aspect ratio 16:9.
Yes, you may image the chips prior to amplification, please refer to the “Imaging Crystal Droplets preOrpost PCR” protocol for instructions.
Yes, you can recover droplets after PCR. Please refer to the “Recovering Droplets From Sapphire Chips” protocol for instructions.
You may use EvaGreen, please refer to the “Using EvaGreen” protocol for detailed instructions. Please make sure to use the correct analysis configuration file associated with this protocol.
No. SYBR Green use is not compatible with the Sapphire chips. Please use EvaGreen instead.
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.
We recommend using 1 uM final concentration for primers as a starting concentration for probe-based detection and 200 nM for EvaGreen-based detection.
We recommend using 250 nM final concentration for probes.
Yes, you may perform WGA in droplets, which has been shown to improve coverage of sequencing. The application note and final protocol are in preparation, please contact support@stilla.fr for more information.
Our assays are calibrated with the PerfeCTa PCR mixes (Quanta Bio), hence we highly recommend their use. Please note that except for the PerfeCTa PCR mixes, we have not perform extensive calibration so we won’t be able to provide you information about the size of the droplets, reproducibility…
Yes, you can perform one-step RT-PCR in droplets using the Quanta Bio qScript XLT One Step RT-qPCR Toughmix (no ROX). Please remember that you need to add fluorescein to the reaction.
Our assays are calibrated with the PerfeCTa PCR mixes (Quanta Bio), hence we highly recommend their use. Please note that except for the PerfeCTa PCR mixes, we have not perform extensive calibration so we won’t be able to provide you information about the size of the droplets, reproducibility…
Our Crystal Digital PCR process is compatible with Quanta Bio PerfeCTa Multiplex qPCR Toughmix (No ROX), PerfeCTa qPCR Toughmix (no ROX) and PerfeCTa qPCR Toughmix UNG (no ROX). Please visit the Quanta Bio website (http://www.quantabio.com) to find distributors in your country.
Yes, fluorescein needs to be added to the PCR or RT-PCR mix for probe-based Crystal Digital PCR experiments. If you wish to use Eva Green for amplicon detection, you need to add Alexa Fluor647 to the mix.
No, we do not recommend that you use ROX as a reference dye. This dye may precipitate in certain conditions in droplets, which hinders software analysis.
First you may prepare a solution at 1mg/ml (2.6 mM) in molecular grade water, which you can then dilute to generate a 100 uM stock solution. Finally, we recommend preparing a working solution at 1 uM, which you can add directly to the PCR mix.
You need to resuspend fluorescein in molecular-grade water.
You may order fluorescein from VWR, catalog name: Fluorescein Sodium Salt, High Purity Grade, Catalog number: 0681-100 g.
Yes. You may use AlexaFluor647 as a reference dye, however you need to use another analysis configuration file, which we will provide. Please contact support@stilla.fr if you wish to change reference channels.
No. The Crystal Miner software needs a basal fluorescent signal to be able to count all droplets, this is why we add fluorescein to the mix.
The fluorescein is added to the PCR mix to provide a basal fluorescence signal for droplet recognition (in the blue detection channel) by the Crystal Miner software.
Fluorescein is added to the PCR/ RT-PCR mix at a final concentration of 100 nM
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