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Hungary's Avidin Combines Single-Cell Digital PCR with Patch Clamp for Neuronal Analyses

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Scientists from Hungarian pharmaceutical development firm Avidin and the Hungarian Academy of Sciences have combined digital PCR on Life Tech's OpenArray platform with patch-clamp recording to enable exact quantification of gene expression and correlate it to phenotypic data in individual neurons, according to research published this month.

The method is useful for better understanding the genetic underpinnings of neurodegenerative diseases at the single-cell level and to identify and define new molecular targets for therapeutics and diagnostics, Avidin CEO László G. Puskás told PCR Insider this week.

The method is also a prime example of the power of single-cell genetic analysis, which is likely to be combined more frequently with digital PCR in the future, Puskás said.

Avidin is an approximately 10-year-old privately owned company focused on developing novel chemical entities that it then seeks to license or sell to big pharma to develop as drug candidates. In some cases Avidin also forges collaborations to advance chemical entities though the drug-development process.

The company's primary focus is on cancer and neurodegenerative disease, particularly Alzheimer's disease, which is Avidin's most advanced program, Puskás said.

"We would like to better understand what's going on in an Alzheimer patient's brain, or in, for example, a transgenic mouse model, at the cellular level, the neuronal level, and to define new molecular targets for therapeutics and diagnostics," Puskás said. "This is how we can use this technology that we optimized."

Prior to developing the single-cell digital PCR-patch clamping method, scientists at the company would "make a statistical readout of messenger RNA expression" in a specific region of brain tissue, Puskás said. "This contains lots of different neuron types … so it's very general and averaged data."

However, Avidin was seeking to focus on a particular subset of neurons that it believed plays an important role in completing neuronal circuits and may be a "major player" in Alzheimer's disease development or progression.

Thus, the group decided to employ patch clamp analysis and standard single-cell qRT-PCR to investigate single cells from the subgroup, but found that qRT-PCR methods fell short when it came to detecting low-level gene expression and rare transcripts.

"For rare transcripts, it is very difficult to quantify with traditional single-cell qRT-PCR," Puskás said. "Sometimes it works, sometimes it doesn't. It matters whether there is 1, 2, 3, 5, or 12 copies in the same cell. When you have very small amounts, then the data can be distorted very much using traditional technology."

Their solution, which is described in a paper published in the June issue of BioTechniques, involves quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using "high-density nanocapillary digital PCR."

More specifically, the method entails conducting patch-clamping of single neurons using a micropipette, then aspirating as much of the intracellular content as possible into the recording pipette by applying a gentle negative pressure while maintaining a tight seal.

The contents of the pipette are then expelled into a test tube and snap frozen or immediately used in the PCR step.

With standard single-cell PCR, the researchers would then extract and purify nucleic acids, combine this with specific targeted probes and master mix, then run the amplification reaction. But with digital PCR, the researchers instead evenly divided the reaction mixture into discrete nanoliter-scale reaction volumes on Life Tech's OpenArray system, thermocycled, and used the OpenArray software to analyze each well in an attempt to locate specifically targeted amplification products. By doing so, the researchers are able to increase the detection sensitivity of specific DNA molecules by increasing the target-to-background ratios.

The researchers used their method to quantify expression of a known cell-type dependent marker gene, gabrd, as well as oxidative-stress-related induction of hspb1 and hmox1 expression, in individual neurogliaform and pyramidal cells, respectively.

They found that the miRNA mir-132, which plays a role in neurodevelopment, was equally expressed in three different types of neurons. They also further validated the accuracy and sensitivity of their method by using synthetic spike-in templates and detecting genes with very low levels of expression.

Not only does the method allow Avidin researchers to correlate specific physiological activity from single cells with specific gene expression activity, but it may also enable them to investigate how a drug candidate might alter this correlation.

"We would like to apply our drug candidates for Alzheimer's disease," Puskás said. "We know it acts at multiple targets. But exactly how it works, we don't know. It would be nice to deconvolve the effects of our drug at the single-cell level. It means we can apply different [chemical entities] in a brain slice just near a specific neuron and can detect how it behaves specifically to a certain insult. So we don't have to, for example, make ischemic or hypoxic threats to the entire brain — just to certain cells."

So far, Avidin has used the technique to narrow in on an "extremely exciting gene that was functionally active in one specific type of neuron" and may be a promising target for Alzheimer's drugs. Puskás declined to provide additional details because the company is currently hoping to publish a peer-reviewed paper describing these findings.

Puskás also told PCR Insider that the company has purchased two additional digital PCR systems — Bio-Rad's QX100 and Life Tech's QuantStudio 12K Flex — to potentially combine with its single-cell patch clamping/digital PCR method.

Both of the technologies, Puskás said, are designed more for low copy number detection as opposed to low-level gene expression. "Those are mainly for screening [thousands] of messages and fishing out one mutant message."

The BioTrove platform is not optimal for that purpose, but is ideal for single-cell gene expression studies. In addition, Avidin has maintained a "very good connection and long history" with Life Tech and prior to that BioTrove, which developed the OpenArray technology but was later acquired by Life Tech.

Puskás said that the company is open to exploring all digital PCR techniques that emerge, and especially single-cell-oriented platforms. "I think there will be more combinations of single-cell harvesting and digital PCR, and that's an interesting merge," he said.

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