By Ben Butkus
This article was originally published on April 20.
NanoString Technologies this week unveiled a new product for microRNA analysis that uses multiplex digital barcode technology to profile the human miRNA transcriptome in a single tube.
In addition, NanoString disclosed that researchers from the University Health Network in Toronto, the Mayo Clinic, Ohio State University, and University of Miami School of Medicine are among the early-access customers who have been using the product ahead of its commercial launch.
The new human miRNA expression assay kit is based on the same digital barcoding technology that forms the foundation of NanoString's gene expression CodeSets and, as such, can be run on the company's nCounter prep station and digital analyzer.
The company's gene expression CodeSets are pairs of approximately 50-base probes that hybridize and are used to detect and count mRNA transcripts. The nCounter digital analyzer collects data from multiple probe complexes, each one immobilized in color-coded cartridges.
According to NanoString, the technology can detect and count hundreds of gene transcripts simultaneously with a sensitivity of less than one copy per cell. As such, the company has been positioning the nCounter gene expression assays as bridging a gap between qPCR and microarrays.
Sean Ferree, director of product development for NanoString, told PCR Insider that the company developed the new miRNA expression assay kits partly because existing users of the mRNA expression kits had asked for it; and partly because NanoString's scientists realized that miRNA expression would be a good fit for the nCounter technology.
"There [has been] a pretty large growth rate in miRNA research in the last few years," Ferree said. "There are a number of applications out there for profiling miRNAs. But our team did also identify miRNAs as a very good fit for our technology because … it has multiplexing capabilities in the hundreds of different species."
That fits well with miRNA profiling, Ferree added, because the current version of the Sanger miRNA database contains several hundred human miRNAs.
"Also, unlike mRNA expression, where our technology is very well-suited to validating and routine testing of whole-genome screens, with miRNAs we are … also able to do discovery experiments because we can cover the entire micronome," Ferree said.
NanoString is also touting the simplicity of the miRNA expression assays because they are only slightly modified versions of the already proven mRNA assay kits.
"Our mRNA expression technology uses two 50-base target-specific probes, and miRNAs are 22 bases in length, so it's pretty obvious that our standard expression approach is not going to work to target miRNAs directly," Ferree said.
To get around this problem, the company incorporated a target-specific ligation step, in which a specific DNA oligonucleotide of around 70 base pairs is designed for every microRNA. The user then "takes that 70-base oligo, and does a specific ligation to a specific miRNA. This is a multiplex ligation, so we put hundreds of different DNA oligos into solution with the miRNA sample; and then we do splinted ligation, so we have hundreds of different bridges that will splint the miRNA to the oligo."
The end result is an miRNA-DNA chimera that is roughly 100 bases long, which becomes the target for the standard CodeSets hybridization and analysis step, Ferree said.
"The beauty is we have a new up-front sample processing step, and then everything else uses the same reagents, the same processing, everything as our mRNA assay. So it drops perfectly into the nCounter system," he said.
Overall, NanoString said that the assay kits enable researchers to perform highly multiplexed, direct digital detection and counting of miRNAs at single-base resolution without the need for PCR amplification. Further, researchers can profile more than 700 human and human-viral miRNAs with specificity and sensitivity comparable to qPCR at a fraction of the cost, the company said.
Ferree said that there are not currently any published studies available to support these claims, but that the company is "currently running those studies with a number of collaborators, and we hope to have that publicly available in the near future."
NanoString unveiled the new kits at the American Association for Cancer Research annual meeting this week in Washington, DC. The kits will be ready for shipment in a couple of weeks, Ferree said, so the goal of the AACR launch was to drum up some orders in advance of availability.
A number of academic collaborators have been testing the kits in advance of their launch. One of those early-access customers, Neil Winegarden, head of operations for the University Health Network Microarray Centre in Toronto, told PCR Insider this week that it sees the new kits as a supplement to qPCR in some cases, and a replacement in others.
"What initially attracted us to the platform is that we've been doing a lot of high-content discovery-based work with microarrays, and one of the problems that our customers always faced is [finding] a good way to validate those hits," Winegarden said.
A given microarray experiment might produce hundreds of hits, Winegarden said, and "typically people have validated those using qPCR, but they'll take a dozen hits, validate those, and hope that what they found for that dozen holds true for the rest of the hits in the panel, and we know that's absolutely not true."
"What we liked about the NanoString platform was the ability to validate the whole lot," he added. "Also, one of the reasons we like this over qPCR is that every single gene is measured at exactly the same time, but in individual reactions. For me, the gene-to-gene bias is greatly reduced."
The microarray center, which is akin to a core facility, plans to offer the miRNA assays as a service to all of its customers at large, Winegarden said.
"If you've only got two, three, or a half-dozen things to validate, qPCR is probably still the best way to do it," he said. "But if you've got dozens that you want to look at, then to me, NanoString really takes over."
Other early-access users include researchers at the Mayo Clinic Comprehensive Cancer Center, Ohio State University Comprehensive Cancer Center Nucleic Acids Shared Resource, and the Oncogenomics Core Facility at the University of Miami School of Medicine's Sylvester Cancer Center.
In a statement, Carlos Croce, an miRNA researcher at OSU, called the new kits "a true breakthrough" in miRNA research tools. "Researchers now have a powerful tool to study miRNAs in cancer and other disease states, as well as to develop potential diagnostic and therapeutic strategies to mitigate the effects of abnormal miRNA expression," he added.