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Fluidigm Developing Assay Panels for Gene Expression Studies in Single Stem Cells

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By Ben Butkus

Fluidigm has developed a new version of its microfluidic Dynamic Array integrated fluidic circuits pre-loaded with a panel of stem cell genes that will allow researchers to conduct PCR-based gene expression studies on single stem cells, company scientists disclosed this week.

Fluidigm is now seeking stem cell researchers with which to collaborate to beta-test and provide feedback on the gene expression panels prior to an anticipated broad product launch later this year, the scientists said.

If successful, Fluidigm plans to follow the stem cell panels with gene expression panels for interrogating a variety of genes involved in cellular metabolism and regulation, as well as for microRNA expression, in single cells.

The stem cell gene panels will also be the first time that Fluidigm – traditionally a manufacturer of microfluidic chips and instrumentation – will offer pre-tested assays specifically designed for use on its those products, according to a company representative.

"We're really trying to take away the tedious parts of the research to allow scientists to do the important work," Fluidigm spokesperson Howard High told PCR Insider this week.

In an interview with PCR Insider, High, Martin Pieprzyk, a product marketing manager at Fluidigm, and Alan Mir, a scientist in the company's R&D group, discussed the company's foray into single-cell biology and its decision to develop assay content specifically for stem cell research.

"Fluidigm has designed all of its platforms to be open," Pieprzyk said. "Unlike microarrays or any of our major competitors who pre-package content with their chips, we kind of leave it up to our customers to decide what kind of chemistry, what genes, or what SNPs they want to look at."

About four years ago, Fluidigm introduced a protocol for researchers to conduct single-cell gene expression profiling on the company's Dynamic Array chips and Biomark real-time PCR instrument.

The decision to do so was spurred partly by the increasing popularity of single-cell studies to account for heterogeneous gene expression in certain cell populations such as tumors cells; and partly due to the fact that Fluidigm's microfluidic technology is well-suited to low-volume applications such as single-cell analysis.

Fluidigm originally targeted the single-cell protocol at customers conducting oncology research, and many of them adopted the protocol for their studies, Pieprzyk said.

"Then we started to market it to some stem cell researchers, and we found very quickly that in the stem cell world, the ability to look at single cells is even more important than it is in cancer," Pieprzyk said. "That's when the protocol really took off."

Since then, he said, "many of the top stem cell researchers in the world have either used our system or have one of them," and use it for single-cell gene expression for stem cell research.

"That's all fine and good, but the reality is, for this to be appealing to the broader market, for people that are not quite on the cutting edge; that aren't willing to go figure out their gene panels for each experiment; and that have a very clear experiment in mind, but would like a starter gene panel, or a starter kit – for those people, until now, we have not really had a good solution," Pieprzyk said.

Fluidigm's first pre-designed panel, a stem cell panel, consists of 160 genes of interest and control genes based on in-house research, published literature, and publicly available information. "Basically we will provide you with this pre-developed content that will allow you to look at your stem cells against these key 160 stem cell genes, from the get-go, without additional work on your part," Pieprzyk said. "Then as you gain more data and experience, we are still an open platform, which means that if you need to substitute or add genes, we can do that."

Mir told PCR Insider that the protocol for the single stem cell gene-expression assays will be similar to other single-cell protocols on the company's Dynamic Array chips.

"We spot individual cells in a 96-well plate, sort of a standard methodology in about 5 microliters of solution," Mir said. "In that solution they can be lysed and frozen and stored for several months. Then we have something called specific transcript amplification, where we throw in a panel of primers that are specific for the genes you want to look at, and then we amplify.

"Then we take an aliquot of that amplification, and do a very standard gene expression assay that Fluidigm does all the time," he added. The 96.96 Dynamic Array chip technology allows researchers to assay 96 individual cells against 96 individual genes at the same time.

Mir said that Fluidigm and its customers have been performing the amplification with Taqman reagents. "The protocol is not rocket science, but it works," he said. "Taqman's great, and we have an option to use it," but Fluidigm instead prefers to use EvaGreen, a SYBR Green alternative dye produced by Biotium. "We would design an inexpensive, completely customizable EvaGreen gene expression panel for stem cell-related genes. We're looking at 160 now, but that number can change depending on customer interest."

Mir also stressed that the content would eventually be "completely customizable" and inexpensive, which is "a consequence in part of a low cost per assay from using the Fluidigm chip, and also because we're going to be offering these as a DNA-binding dye approach as opposed to using Taqman. So the cost is trivial."

Mir and Pieprzyk said that the stem cell panel has been developed in house and "is ready to go; we have data for it, we know it works well … but what we need now are stem cell collaborators – people that are doing lots of stem cell work … to take the panel and begin to run it, and actually gain real data from it, and give us feedback on how the genes we selected are working; what genes they would like to add; and what is the biological significance of some of the genes.

"Basically, we want researchers to do a full-on beta test site run before we take that panel to the real world," ideally within a few months, Pieprzyk said.

And although Fluidigm's first round of assays will be stem-cell related, the company also has in the pipeline "a whole range of panels for general metabolism such as cell cycle, growth, cytokines; essentially the panels of biochemical genes that people have been looking at for some time, but we can completely customize it," Mir said.

Further, like many companies in the genomic research tool space, Fluidigm is also eyeing microRNAs. "Now we have a protocol that allows a researcher to look at microRNAs from individual cells, and look at the profile of potentially all the known human microRNAs," Mir said. "There are about 640 of them right now, and in theory we could look at all of the microRNAs from individual cells for a variety of applications."

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