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Affymetrix Targets Single-cell Analysis Field with Launch of New PrimeFlow RNA Assay

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NEW YORK (GenomeWeb) ― Affymetrix has introduced a new flow cytometry assay capable of detecting RNA and proteins at single-cell resolution.

Developed within Affymetrix's eBioscience business, the PrimeFlow RNA Assay allows users to profile RNA and protein expression within millions of single cells while conventional flow cytometry approaches remain limited to antibody-based analysis of cell surface and intracellular proteins.

Given these capabilities, the assay "has great potential both for the advancement of research and for providing revenue growth," Dylan Malayter, senior product manager for the company's eBioscience business, told GenomeWeb.

"We see growth opportunity with our core target market of immunologists, stem cell biologists, and oncologists, and see the assay as one of the many opportunities we have to participate in the single-cell analysis market," Malayter said.

Affymetrix has been targeting the single-cell analysis market more aggressively in recent years. In February, CEO Frank Witney cited the market as one of the Santa Clara, Calif.-based company's major growth opportunities. In a statement last week, Witney said the new assay showcases "one of the many exciting opportunities we have for integrating cell analysis and genomics, which open up new avenues for innovation and growth."

For Affymetrix, perhaps best known for its microarray products, the new assay combines capabilities gained through its acquisitions of Panomics and eBioscience in 2008 and 2012, respectively. Specifically, it marries Panomics' QuantiGene ViewRNA fluorescence in situ hybridization assay for RNA detection with eBioscience's flow cytometry reagents. 

It also builds on an existing assay. Last year, the company introduced QuantiGene FlowRNA, which allows the measurement of up to three RNA transcripts in a single cell, in addition to intracellular protein targets, cytokines, and transcription factors. The assay paired oligonucleotide probe design with branched DNA signal amplification to enable gene expression profiling at the single-cell level.

A paper describing the use of QuantiGene FlowRNA to measure the expression of genes linked to immune cells appeared earlier this month in Nature Communications.

According to Affymetrix, while the new PrimeFlow RNA Assay relies on the same technology, it differs from the older QuantiGene FlowRNA assay in a number of ways. PrimeFlow has a simplified pretreatment protocol, has been enabled for intracellular antibody staining, and has expanded total antibody staining, as it relies on traditional fluorochrome chemicals rather than methanol. Unlike QuantiGene FlowRNA, Affymetrix's new PrimeFlow RNA Assay is packaged with a buffer system that contains either mouse or human species positive controls.

"One of the biggest challenges of [developing] this assay was to further improve [its] utility by enabling the detection of intracellular protein targets, cytokines, and transcription factors, and by expanding the fluorochrome compatibility, all with maintaining the same level of high sensitivity RNA detection," said Malayter. "This was accomplished in [the] PrimeFlow RNA Assay by redesigning the fixation and permeabilization buffers and protocols," he said.

When asked about the assay's applicability versus other approaches, including microarrays and sequencing, Malayter described PrimeFlow as both a complementary approach as well as an assay capable of delivering novel information.

"We have seen great advances in omic techniques that have enabled researchers to identify the most relevant targets in disease research areas," said Malayter. "However, there is still an unmet need for assays with high resolution and high cell input to unravel the relevant targets' regulations and effects at a systems level in complex biology processes," he said.

The "revolutionary" aspect of PrimeFlow is its ability to observe the dynamics of RNA and protein expression within individual cells, according to Malayter.

"It will now be possible to correlate transcriptional events and translation events in the same cells, rather than trying to extrapolate meaning from data from two independent assays, one for RNA and one for protein," he said. "Microarrays and sequencing can provide comprehensive gene expression data in bulk sample preparations or extremely limited number of cells [but] the analysis of bulk samples can mask the individual effects of unique cellular subsets," he added.

Affymetrix is interested in expanding the applications of PrimeFlow, Malayter noted. Soon, customers may be able to run the assay on samples directly from whole blood after lysis, "enabling faster sample preps across donors." The company is also looking at ways to support the analysis of greater numbers of samples, moving from individual tubes to 96-well plates, Malayter said.

Though Affymetrix has just launched PrimeFlow, the assay already has a number of proponents. Among them is Filippos Porichis, director of international programs at the Ragon Institute MGH, MIT and Harvard, and a co-author on the new Nature Communications paper.

In the paper, the authors described optimized protocols that rely on the assay to detect mRNA and microRNA in human leukocytes, demonstrating antigen-specific upregulation of  several mRNA targets in HIV- and CMV-specific T cells. They also showed simultaneous detection of cytokine mRNA and its corresponding protein in single cells.

For Porichis, the new assay is especially relevant in the field of single-cell analysis, overcoming shortcomings associated with both conventional flow cytometry and sequencing.

"Antibody-based characterization of cells with flow cytometry has been widely used by immunology laboratories for many years," Porichis told GenomeWeb this week. "Unfortunately, there are many proteins for which no specific antibodies are available, and therefore investigation of many markers has been impossible to be achieved with flow cytometry. "

And while RNA sequencing has allowed for the transcriptional analysis of such markers at the single-cell level, such techniques require "specialized skills and laborious methods such as single-cell sorting," he said.

The PrimeFlow RNA Assay in contrast "combines a user-friendly protocol, which has many similarities with standard antibody-staining procedures, with data acquisition for flow cytometry," Porichis said. "This, in combination with the fact that it provides the opportunity to stain cells for any marker of interest at the transcriptional level, allows the investigation of rare phenotypes for any species," he added, making the assay a "very attractive tool for immunology laboratories performing translational research."

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