By Ben Butkus
RainDance Technologies has launched its digital PCR platform for applications such as rare allele detection and copy number variation analysis.
The platform, called the RainDrop digital PCR system, will immediately compete with existing commercial digital PCR products from Bio-Rad, Fluidigm, and Life Technologies — although RainDrop will offer a dynamic range that is "orders of magnitude" greater than rival systems, the company said this week.
In addition, RainDance believes that RainDrop will compete favorably on assay cost — in some cases 100- to 10,000-fold cheaper on a "per-data-point basis," the company said.
RainDance will officially launch its digital PCR platform at the American Association for Cancer Research annual meeting beginning this weekend in Chicago. Andy Watson, RainDance's chief marketing officer, discussed the system's launch with PCR Insider this week.
"We're taking orders now," Watson said. "We're in the middle of recruiting folks that want to be part of our first access program. We'll be giving a dozen or so customers the opportunity to come into our lab and … use the technology on a few dozen of their samples to get them excited and start them publishing … and shortly after that we'll start to ship the instruments out to folks in the field."
In digital PCR, dilute samples are divided into many separate quantitative PCR reaction volumes, which distributes target DNA molecules among the reaction volumes such that the vast majority contains either one or zero target DNA molecules.
Generally, a digital PCR reader then counts the number of "positive" reactions denoted by one fluorophore color and compares it to the number of "negative" reactions denoted by another color or a lack of fluorescence. Extrapolating these numbers provides a quantitative readout of the amount of specific target DNA and normal, background DNA that was present in the original sample.
The technique thus has far greater sensitivity than conventional qPCR, enabling the detection of rare variants in a high amount of background DNA for applications such as rare allele detection, copy number variation, and, potentially, diagnostics.
RainDance's digital PCR platform is powered primarily by the company's picoliter-scale droplet generation and sorting technology. The company already has products on the market that incorporate this technology for targeted resequencing and sequence enrichment applications.
RainDance first disclosed in July 2010 that it was looking into the digital PCR space, noting that its picodroplet technology, called RainStorm, could easily generate the massive number of individual reaction chambers needed to run a digital PCR experiment with a high dynamic range (PCR Insider, 7/8/2010).
Then, in September, CEO Roopom Banerjee provided an update on the platform's commercialization to investors at the UBS Global Life Sciences Conference in New York, noting that the company would aim for a launch around AACR and that the system would likely have the ability to generate around 10 million data points per sample as opposed to the approximately 1 million it originally promised (PCR Insider, 9/22/11).
True to its word, RainDance will launch RainDrop in the coming week with a multiplexed approach that generates between 1 million and 10 million picodroplets in each of eight "lanes," which the company claims is a 500- to 10,000-fold improvement over existing methods. The upshot is the ability to detect one mutant allele among 250,000 wild-type molecules with a lower limit of detection of one in more than a million.
"It's kind of like pixels on a camera," Watson said. "A lot of older cameras didn't have enough pixels to take very good pictures. That's what many existing digital PCR products are like. And really what we're launching is kind of like going from a 300-by-300 black-and-white digital camera to the high resolutions you see today in digital cameras. Digital [PCR] is all about pixels — and we have the capability of generating … enough pixels or enough data points to allow you to get true digital answers."
Watson said that the RainDrop system comprises two separate instruments: the RainDrop "Source," which creates and collects the picoliter reaction volumes; and the RainDrop "Sense," a cytometer-like device that identifies and counts each picodroplet. Users would also need a thermal cycler, which nearly every molecular biology research lab already has.
The Source module, Watson explained, would likely be placed in a laboratory's "pre-PCR" area. Researchers then take samples from the droplet generator, "put them into regular thermal cycling plates or tubes, and then put them on their own thermal cycler."
Following PCR amplification, users would then place the samples into the Sense module, "which basically identifies droplets that have given a positive reaction … and identifies what type of reaction has occurred in there. It's both the identification and the counting that gives us this digital multiplexing capability," Watson said.
In addition, RainDrop is a "closed system, designed so there is no contamination or carryover," he added. "Customers are used to working with closed real-time PCR, and we thought it would be silly to ask them to take a step backward and use an open-tube method with digital PCR, especially when they're looking to get very sensitive results, and even just getting a few droplets from one well into another would confound those results."
Each benchtop instrument would have its own PC workstation, and RainDrop will feature dedicated data-analysis software.
Of the competing platforms on the market, RainDrop most closely correlates with Bio-Rad's QX100 Droplet Digital PCR system, which the company added to its product portfolio with its $162 million October acquisition of privately held QuantaLife (PCR Insider, 10/6/2011).
The QX100 also comprises a droplet generator that can partition a nucleic acid sample into approximately 20,000 nanoliter-sized droplets; and a droplet reader that features two-color fluorescence detector and can count individual droplets at a rate of about 1,000 per second. Bio-Rad has not made specific claims about the platform's sensitivity, but last year, Fred Hutchinson Cancer Research Center scientist Jason Bielas — an early-access user of the QuantaLife system — said that that his lab measures rare mutational events with frequencies of as small as one in 109 in nuclear and mitochondrial DNA, and is able to subsequently conduct single-molecule sequencing on recovered mutants (PCR Insider, 6/30/2011).
QuantaLife was initially selling the platform for around $50,000, although Bio-Rad does not currently disclose the platform's list price. Watson said that RainDrop will cost in the ballpark of $100,000, but added that the system will be very competitive in terms of cost per data point due to the sheer amount of data it produces.
"We look at the running cost … kind of like the DNA sequencing world, where they really think about the cost per gigabase," Watson said. "So the way we like to think about it is in terms of cost per million droplets. Comparing to some of the other droplet platforms, and the microfluidic well platforms, we're 100- to 10,000-fold cheaper on a fundamental per-data-point basis."
Two other major competitors are Fluidigm, which sells the Digital Array; and Life Technologies, which sells the OpenArray. Both products use different means to achieve more or less the same end: a set number of individual PCR reactions — somewhere in the range of tens of thousands — on a physical consumable chip or array.
However, Life Tech at least has argued that the number of individual reaction volumes generated by a platform like RainDance's or QuantaLife's may be "overkill" for most practical laboratory applications. Life Tech in October launched a product intended to distinguish itself from others — the QuantStudio 12K Flex, which allows users to perform both low-throughput and high-throughput quantitative and digital PCR on the same sample using the same software interface (PCR Insider, 10/13/2011).
Meantime, Fluidigm last July licensed from the University of British Columbia a chip-based digital PCR technology with a 100-fold greater reaction chamber density than Fluidigm's current microfluidic valve-based devices, but it has yet to integrate that technology into a marketed product (PCR Insider, 7/14/2011).
At any rate, all the platforms have a similar application sweet spot, which is detecting rare genetic events in a huge background of wild-type noise.
"Customers are using this technology for expression or copy number detection and just wanting to do it better," Watson said. "And second, it's folks who really want to do this low-frequency allele detection in cancer and really have confidence that they can put in a whole range of sample types and detect the number of mutant alleles."
To wit, two of RainDance's early-access users are Pierre Laurent-Puig, a professor of oncology; and Valérie Taly, group leader/CNRS researcher, both at Université Paris Descartes. Laurent-Puig and Taly will be at AACR next week presenting data they have generated using the RainDrop for rare allele detection in cancer.
Last year, Laurent-Puig, Taly, and colleagues at both Université Paris Descartes and Université de Strasbourg published a pair of publications demonstrating new methods and applications for RainDance's digital PCR technology (PCR Insider, 5/26/2011).
The first paper described a digital multiplexing method based on fluorescence color and intensity, and used the method to develop a five-plex assay for spinal muscular atrophy using just two fluorophores. The second paper described the use of RainDance's digital PCR technology to detect a single mutated KRAS oncogene in a 200,000-fold background of normal KRAS genes expressed by cell lines; and used the multiplexing method to screen the six common mutations in a particular KRAS codon in parallel in a single experiment.
Taly told PCR Insider in an e-mail this week that the group now aims to validate its KRAS assay in patient samples, since previous work involved the use of genomic DNA extracted from cancer cell lines.
"This work is basically a first validation of the procedure for [detecting] the duplex (wild-type/one mutation) or multiplex (the seven more frequent mutations of KRAS and wild-type sequences) [in] circulating tumoral DNA within plasma of patients with metastatic colorectal cancer," Taly said. "More precisely, instead of using cell line DNA we have been using DNA extracted from the plasma of patients with metastatic colorectal cancer for whom the mutational status of their tumor has been determined previously by classic procedures."
Watson said that RainDance also has collaborations in place with "a couple of major diagnostic companies" to conduct "feasibility testing" on various assays for low-frequency allele detection; however, he declined to disclose their names due to confidentiality agreements.
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