Scientists from Arizona State University's Biodesign Institute are using a two-year, approximately $420,000 grant from the National Institutes of Health to further develop a tool that combines laser-based excision and ablation with emulsion-based droplet PCR to profile mRNA expression in single cells.
Once developed, the platform could provide life science researchers with a tool to better elucidate cellular heterogeneity in, for instance, cancerous tissue, by maintaining spatial and temporal resolution of gene expression in single cells.
Deirdre Meldrum, a senior scientist at ASU and director of the Center for Biosignatures Discovery Automation at ASU's Biodesign Institute, is serving as principal investigator on the grant, which is being administered by the National Cancer Institute.
Meldrum told PCR Insider this week that her research team began developing its platform as part of prior work it had done analyzing the transcriptomes and metabolism of single cells in conditions such as Barrett's esophagus and inflammation.
"We've been doing single-cell analysis, which is very important because of the heterogeneity in cells," Meldrum said. "We want to also get to more of an environment that is representative of the cells in their natural state … so we'd also like to be able to analyze the cells in situ."
Meldrum added that most current technologies designed for transcriptome analysis either provide a composite picture of cellular gene expression or are limited to disassociated cells so they don't capture true cellular response. In addition, preparing samples for quantitative real-time PCR can take hours or days.
"With this proposal, we will develop a method that will make it possible to actually lyse the cells in situ from a tissue … prepare the cell for qRT-PCR analysis … and go from tissue to output in about an hour," Meldrum said.
The team's technology uses a two-photon laser to serially lyse individual cells at known coordinates within a 3D tissue sample. Two-photon lasers differ from conventional lasers in that they rely on a nonlinear interaction between an ultrafast pulsed light source and biological material to achieve energy transfer to the cell at a precise nanometer-scale focal volume.
"The idea would be that you would be able to, using microscopy, select the cells on the surface of the tissue, and very precisely lyse and analyze cells of interest at the sub-cellular level … and not have contamination from one cell to another," Meldrum said.
Then, the cells are ablated and transported within seconds to microliter-scale emulsion-based droplets for qRT-PCR-based mRNA expression analysis. Carryover contamination between sequentially lysed cells is minimized by optimizing laser power and using hydrodynamic flow focusing with precise flow rate control.
The droplet-based qRT-PCR system is not an existing commercial model such as those offered by Bio-Rad or RainDance Technologies. Rather, Meldrum said, the group has built its own platform in house, and is attempting to patent it along with its single-cell analysis technique.
"It's the same idea [as digital PCR], as the sample … [goes into] tens or hundreds of discrete droplets with very small volumes," Meldrum said. "But we'll have a continuous stream flowing, and that then keeps our temporal information as to when we lyse the cells from the tissue. We very carefully spatially and temporally keep track of what's going on with each sample."
The group noted that its tool should be well-suited to basic biomedical research and clinical applications such as assessing tumor cell population heterogeneity.
As an example, Meldrum cited research her group has been conducting on Barrett's esophagus with researchers from Fred Hutchinson Cancer Center. The partners have previously used fluorescence in situ hybridization to demonstrate the heterogeneity of cells lining the crypt of the esophagus in the disease.
"We now have a nice map showing the cellular heterogeneity," Meldrum said. "Now with this we'll be able to look at the cells lining the crypt and look at the gene expression of those different cells to see how those change. We'll quantitatively determine things like p53 and p16 [gene expression] and other things in the cells."
Other research groups are attempting to address this issue using different means. For instance, researchers from the University of Southern California were recently awarded a similar grant from the NIH to develop a high-throughput, single-cell PCR method to analyze genetic heterogeneity in tissue samples. That group's method uses photolithography to design chips containing a matrix of millions of microwells that can be placed directly onto morphologically intact tissue slices. Then, the researchers perform individual allele-specific endpoint PCR reactions in each of the microwells, which are small enough that they contain only a handful of cells (PCR Insider, 10/4/2012).
"There should be of a lot of interest in this because it does enable you to go right from the tissue to the gene expression, so that you don't lose the RNA or have time for it to change, so it will be more truly representative of what's going on in a tumor," Meldrum said.
Like the USC scientists, the ASU researchers are considering routes to commercialize their technology, with recently filed patent applications the first step.
In the meantime, they will use their NIH award, which began in late September, to further develop and test each component of their workflow and execute proof of concept studies.
"It's a pretty fast development path, a two-year project, and there's a lot more that needs to be done — optimizing the process for different kinds of cells present in a tumor; optimizing the thermal cycling; the whole microfluidic aspect; integrating all the different parts so that you can truly go from tissue to output in about an hour," Meldrum said.