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Australian Lab Uses Sound to Improve Reverse Transcription in Single-Cell Gene Expression Studies

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By Ben Butkus

Australian scientists may soon be cranking up the music in their labs in order to improve the efficiency of their single-cell gene expression studies, thanks to a method developed by researchers from the University of Melbourne.

The technique, called acoustic microstreaming, improves cDNA yields from reverse transcription of single-cell quantities of RNA 10- to 100-fold over standard methods by promoting better reagent mixing — or "micromixing" — in standard PCR tubes, the researchers said.

In addition, the group has applied for patent protection on its method and is currently in discussions with Australian laboratory instrument manufacturers to further develop and commercialize the technology.

In an e-mail this week to PCR Insider, Tim Aumann, an investigator at the University of Melbourne's Florey Neuroscience Institutes, discussed how his research group invented the micromixing method as part of its research on correlating gene expression of single maturing neurons with their electrophysiological properties.

"Following electrophysiological characterization of each cell, a small sample [of about 20 percent] of the cell’s cytoplasm was aspirated into the recording micropipette for RT-PCR analysis of its gene expression profile," Aumann explained.

"Using standard laboratory RT-PCR methods, we were frustrated by the small number of genes we could examine per cell; the qualitative nature of the readout; and the inability to detect low-abundance transcripts," Aumann said.

Aumann noted that many labs have resorted to microfluidic approaches to increase the yield of cDNA from reverse transcriptase reactions of single-cell amounts of input mRNA. Indeed, companies such as Fluidigm, which offers microfluidic chips for such applications, have carved out a unique space in the single-cell gene expression market.

However, Aumann claimed that such technology is not yet widely available and that the approach is costly and requires specialized equipment.

"We reasoned that the success of microfluidics was due in large part to increasing the rates of interactions between [reverse transcription] reagents within the much smaller reaction volumes," typically nanoliters, Aumann said. "Thus, if we could more efficiently mix RT reactions in standard laboratory volumes [of microliters], we should also see improvement."

In order to test their hypothesis, Aumann's group contacted colleagues at the Commonwealth Scientific and Industrial Research Organisation, who had recently filed a US patent application on an acoustic microstreaming method designed to dramatically increase the rate mixing of two or more solutions in microliter volumes.

Aumann said that the relatively simple setup for acoustic microstreaming essentially involves placing a pair of small, loud computer speakers underneath a plate into which standard Eppendorf PCR tubes containing the reverse transcription reagents and mRNA have been placed.

Reporting in the February edition of Biotechniques, the researchers tested the method on RNA isolated from neurons in adult mouse brain slices. They performed RT reactions with RNA concentrations ranging from 1 nanogram/microliter to 0.1 picograms/microliter, with the lower end of this range representing a single-cell equivalent amount of RNA.

They also employed two different mixing protocols: micromixing for only the initial five minutes of a 60-minute reaction; and micromixing throughout the entire reaction. They assessed cDNA yield from the RT reactions by performing qPCR using primers designed to amplify two low-abundance test genes expressed by dopaminergic neurons of the midbrain: housekeeping gene Hprt and transcription factor Nurr1.

Indeed, micromixing significantly decreased the number of qPCR cycles needed to detect cDNA representing Hprt and Nurr1 by about nine and 15 cycles, respectively. According to the researchers, this is equivalent to performing reverse transcription with between 10- and 100-fold more cDNA in the absence of micromixing.

However, the researchers found that micromixing had a negligible effect on RT reactions comprising relatively high concentrations of input mRNA, a result they chalked up to the idea that at high concentrations, diffusion alone is sufficient to optimally mix the reagents.

Further exploring this notion, the researchers conducted highly sensitive fluorescence imaging of both low- and high-concentration RT reactions to visualize the micromixing process and distinguish it from standard diffusion processes; thereby lending additional support to the idea that micromixing was the main reason for the cDNA yield improvement.

"The most surprising finding was the magnitude of the increase in RT reaction efficiency at the single-cell level," Aumann said. "This should be enough to dramatically increase the number of assessable genes in single-cell samples, enable quantitative assessment of these genes, and enable detection of low-abundance transcripts. Indeed, we found acoustic microstreaming enabled reliable detection of an otherwise undetectable transcript."

Aumann also noted that the improvements in cDNA yield were "over and above what could be achieved using standard methods of mixing, such as shaking, vortexing, or trituration."

The researchers have applied for a patent covering the micromixing method, and are currently in discussions with potential industrial partners, including Australian laboratory equipment manufacturers Scientifix and Dynamica, Aumann said. "We are hopeful that the technology will be commercially available in the near future," he said.

In the meantime, the group will be applying the technique to its own research on better understanding dopamine neurogenesis in the adult brain. "The application of acoustic microstreaming to RT reactions comprising single-cell samples should dramatically hasten the attainment of this goal," Aumann said.


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

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