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Publications Demonstrate Multiplexing, Clinical Potential of RainDance Digital PCR Tech

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By Ben Butkus

A pair of recently published research papers has demonstrated the high degree of sensitivity, clinical diagnostic potential, and multiplexing capability of digital PCR products under development at RainDance Technologies.

First, in a study published online last week in Lab on a Chip, scientists from RainDance and Université de Strasbourg in France described a new method designed to "break the color barrier" of digital PCR by using a multiplexing technique based on fluorescence color and intensity; and used the method to develop a five-plex assay for spinal muscular atrophy using just two fluorophores.

Meantime, in a second study, also published online last week in Lab on a Chip, scientists from Université de Strasbourg, Université Paris Descartes, and RainDance used the company'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.

According to Andy Watson, RainDance's recently appointed vice president and chief marketing officer, the studies provide "high-quality science" and "great proof-of-concept validation" that RainDance's digital PCR technology has great potential in highly multiplexed rare allele detection, copy number variation assays, and clinical diagnostics.

"This is work that is part of any good product development — the lab scientists working with prototypes to really show how this works in new applications," Watson told PCR Insider this week. "This really gives us confidence [to] ramp up significant product development … and that we're working on a technology that will revolutionize the way quantitative PCR is performed."

Key to both research studies is an ongoing collaboration between RainDance and the laboratory of Andrew Griffiths, a founder of RainDance and director of the Chemical Biology Laboratory at the Institut de Science et d'Ingénierie Supramoléculaires, or ISIS.

In the first research study, Griffiths lab members, along with RainDance employees Darren Link and Jonathan Larson, explained their technique for multiplexing digital PCR, a feat that has not yet been accomplished, according to RainDance's Watson.

In conventional digital PCR, dilute samples are divided into many separate reaction volumes. RainDance accomplishes this using its RainStorm microdroplet-generation technology.

As explained in the paper, doing this distributes target DNA molecules among the reactions such that the vast majority of reaction volumes contain either one or zero target DNA molecules.

After mixing in the appropriate nucleic acid primers and fluorescent probes, researchers can then literally count the number of "positive" reactions denoted by one fluorophore color, and compare it to the number of "negative" reactions denoted by another color. 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.

Although the sensitivity of this approach is much greater than that of conventional qPCR, thus allowing the detection of rare variants in a high amount of background DNA, the technique has still been limited to only three- or four-plex due to the spectral overlap of fluorescent probes.

The technique developed by researchers from RainDance and the Griffiths lab can perform much higher multiplexing due to the use of varying concentrations of fluorescent probes that in turn produce varying fluorescence intensities. Thus, even using a single color fluorescent probe, the technique can discriminate among different reactions based on their fluorescence intensity levels. By also using different colors, the technique can be multiplexed exponentially.

"We just control the amount of the fluorescent probe that goes into each one of the assay droplets," Watson said. "When we're reading out these assay droplets, we're reading 'yes' or 'no.' We're not looking to try and get a quantitative measurement in each droplet."

"It turns out that … we can tune the fluorescence intensity of each positive droplet to be specific for a given assay," Watson added.

Competing digital PCR approaches, such as those offered by Fluidigm, Life Technologies, and QuantaLife, can multiplex based solely on different fluorescent colors, RainDance President and CEO Roopom Banerjee further explained.

"If you have three colors, you have a three-plex; five colors, a five-plex," Banerjee said. "The reason we say this is breaking the color barrier is, for the first time, we can use a … two-color system to do exponential multiplexing. In other words, if we use 10 [fluorescence] intensities, that actually gives us a 10-by-10 optical readout, or 100-plex. If we do five intensities, with two colors, it gives us a five-by-five, or 25-plex. This makes it very scalable. It's a really elegant, simple way of exponentially creating multiplexing capabilities."

In the first Lab on a Chip paper, the researchers demonstrated proof of concept by creating a five-plex TaqMan digital PCR assay for spinal muscular atrophy, using just two fluorophores, to simultaneously measure the copy number of two genes and to genotype a SNP. According to Watson, the group has internally created as high as 50-plex assays.

Although the theoretical limit of this multiplexing capability is very high, the practical limit that is currently potentially useful to customers conducting "clinically relevant research assays" is likely in the 10- to 20-plex range, Banerjee said.

"That said, there are certain gene expression assays that can get up to the 20-, 40-, or even 100-plex levels that are used more for research or validation purposes," Banerjee said. "Our dPCR solution over time will certainly have the ability to meet that need; however, it's frankly not a large enough market segment now where we feel we'd need that on the first day on the market. The fact that we can multiplex at all is a true breakthrough in the dPCR space."

In the second Lab on a Chip paper, members of the Griffiths lab, along with RainDance's Link and Larson and collaborators from Université Paris Descartes, provided the first proof of concept for the use of RainDance's digital PCR technology and TaqMan assays in potential clinical diagnostic applications.

Specifically, the researchers used RainDance's technology to determine the mutant allele specific imbalance of a mutated KRAS gene in several cancer cell lines; and to precisely quantify a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes expressed in engineered cell lines.

According to Watson and Banerjee, this level of sensitivity is due to the ability of RainDance's droplet-generation, -sorting, and –detection technology to analyze a much larger number of exceedingly small reaction volumes than possible with other technologies.

"The number of droplets that is required to get this level of sensitivity in these assays … is really large," Watson said. "If you try to detect something that's there [at a dilution of] one-in-100,000, you need right around 1 million droplets … to get that level of sensitivity. And that’s really what we're seeing in that second paper."

Added Banerjee, "if you look at other competitors in the market, Fluidigm and QuantaLife … are at the nanoliter scale. We operate at picoliter scale, so between 100 and 1,000 times smaller in terms of our droplet size. And second, out of the gates, we routinely run dPCR at between 1 million and 10 million droplets per sample. So it kind of blows away [competing technologies] in terms of scale or magnitude."

RainDance, which has traditionally used its patented picoliter-scale droplet-generation and sorting technology for sequence enrichment and targeted resequencing applications, last year told PCR Insider that it was eyeing the digital PCR space for its technology, but declined to provide specific potential applications or products (PCR Insider, 7/8/2010).

And although the recently published papers provide some clarity regarding the company's fledgling digital PCR play, Watson and Banerjee this week still shied away from providing a specific product-development plan.

"We're not ready to provide a specific timeline yet," Watson said. "But we can say that we are lining up collaborators and have been working hard on product development. So you'll see some news over the next six months [regarding] early-access partners and things like that."

Banerjee added that the company would in the coming weeks be revealing a "collaboration and supply agreement with one of the largest chip providers in the world" to produce on a larger scale the prototype chips fabricated and used by Griffiths and colleagues for the recently published research papers. That undisclosed company, Banerjee said, "will also be providing our digital PCR chips in the long term."

Banerjee also said that RainDance currently is working with several undisclosed "strategic partners" to further assess the digital PCR technology, and that the company has been "pleasantly surprised by both the business development and collaborative interest out of research labs and large diagnostic companies" that it has been in touch with.

"This is a true high-resolution alternative to both qPCR and sequencing, and should be quite durable in the market," Banerjee concluded.


Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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