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SUNY Scientists Developing Single-Cell Droplet Separation for High-Throughput Expression Analysis

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By Molika Ashford

State University of New York researchers are using a two-year grant from the National Institutes of Health to develop a microfluidics method to measure single-cell gene expression by creating cDNA libraries from single cells inside of droplets.

Led by Helmut Strey, a biomedical engineer at SUNY Stony Brook, the team is working on a droplet microfluidics device to extract mRNA from single cells for the potential "continuous analysis of gene expression via PCR at rates of up to 3,000 cells/sec," according to the research group's website.

Strey told PCR Insider this week that droplet microfluidics methods offer an attractive system for single-cell examination. But not all of the steps have been hashed out yet to allow the extraction of mRNA into individual droplets for gene expression analysis on the single-cell level.

"We are using microfluidics to essentially put a single cell per droplet and then process them sequentially. So we lyse the droplets, we extract the mRNA, and the ultimate goal is that we create a complementary DNA library inside a droplet," Strey said.

While the group is not currently working on connecting this separation method with PCR, the ultimate goal is to harness the technique to a quantitative PCR system.

"What I think is exciting about it is that these libraries would be recyclable, so we can reuse them. We could, for example, … probe genes inside these droplets and then recover the library completely. That would allow [us] then to measure gene expression of multiple genes in, let's say, a million cells. And there is no technology that can do this right now," he said.

The two-year NIH grant, administered by the National Human Genome Research Institute, provides $221,000 in funding for the project's first year, which began on June 1. In its grant abstract, the group writes that the method should allow for multi-step reactions involving buffer exchanges, which have thus far been difficult because of a lack of technology to "continuously extract and concentrate molecules of interest within individual droplets."

"Droplet microfluidics in general currently has only the ability to add," Strey said. "You can add to droplets, so you could, for example, add reagents to each other. But so far what was impossible was to subtract from a droplet."

Strey said that the team has already achieved a method that they plan to use to extract mRNA from the complex mixture of molecules inside a cell and separate the mRNA into its own private droplet.

Extraction and concentration will be done, the researchers write in their abstract, by binding mRNA to magnetic microparticles and "splitting droplets along the droplet flow axis under the influence of a magnetic field to separate the part of the droplet that contains microparticles from the part that is devoid of particles."

According to Strey, the group is now testing the microparticle extraction method with cells and mRNA.

Next the group will work on subsequent steps toward creating the cDNA libraries. According to Strey, there are still several hurdles. "Cell lysis for example," he said, "is something people have not done yet really in droplets. That's still an open question of how well can you do this."

He said the group's focus is to complete the project step by step. "We have developed the technology to subtract from droplets, then we have to essentially get cell lysis and cell encapsulation right. Then the next step is to combine these things and then get to cDNA libraries," he said.

Strey expects to have finished the process within two years.

The ability to separate mRNA from a single cell into its own droplet opens many avenues of research, Strey said.

"The main focus I think is that we can look for rare cells," he said. "The idea is not to do a full gene expression analysis of a million cells. That would be very daunting. But one thing that this technology can do is to identify cells that are special, that are different than the main population."

"To pull out droplets that have a different gene expression we could look for certain patterns with this technology," he added. Then if you find [the patterns], you can pull them out and then fully sequence them."

Strey said such a technique would be important to cancer research, monitoring and evaluating cancer treatment, and as a basic science tool to study the mechanism of gene regulation on the single-cell level.

The group hasn't developed the method with a particular PCR platform in mind, Strey said, adding that companies working on harnessing microfluidics for digital PCR, like RainDance Technologies, might be a good fit for the group's separation technology. RainDance's microfluidic droplet technology is capable of running digital PCR at between 1 million and 10 million droplets per sample (PCR Insider 05/26/2011).

However, Strey said the group has not solidified how the process would be coupled with PCR, or which platform would ultimately be best.

"What is exciting," Strey said, "is that a lot of technologies have been developed around these droplets. It’s a hugely growing field. That's nice, because we are working on a different direction, so when we have the cDNA libraries of single cells, then we can look at the landscape and see who has the best technology to do gene expression analysis."

Strey said that the group has submitted a patent application for its separation process, but noted that he is interested primarily in the research aspect and is not focused on any potential commercial prospects for the method at this time.


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

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