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Singapore's MiRxes Spins Out from A*Star with Novel qPCR Assay Platform for microRNA Analysis

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MiRxes, a Singapore-based startup focusing on microRNA research and diagnostics, officially launched earlier this month with a proprietary qPCR platform for identifying microRNA and other small RNA biomarkers.

The platform, called mSMRT-qPCR, enables efficient and accurate identification of circulating miRNA biomarkers using existing qPCR workflows, according to the company, which is promoting its use in applications such as early disease detection and patient stratification, prognosis, and treatment response monitoring.

In addition, MiRxes is a key cog in a recently established public-private partnership in Singapore, spearheaded by the country's Agency for Science, Technology, and Research (A*Star) and designed to identify new disease biomarkers and help speed their transformation into commercial diagnostic products through industrial partnerships.

MiRxes CTO Lihan Zhou and other company officials presented posters describing the mSMRT-qPCR technology and MiRxes' efforts to apply it to the non-invasive early detection of gastric and breast cancers earlier this month at Cambridge Healthtech Institute's Molecular Medicine Tri-Conference in San Francisco.

Though a relatively young field, the idea of using circulating miRNAs as diagnostic biomarkers has gained traction, with the number of publications on the small non-coding RNA molecules ballooning in the past year.

"The reason we are focusing on circulating miRNA is because of the promise of miRNA: they're stable, and they're [accessible] in biofluids — serum, plasma, urine, tears — you name it, you can find [miRNAs]," Zhou told PCR Insider at the conference.

The limited length of miRNAs — 18 to 22 base pairs — and high sequence homology among miRNA family members have made qPCR the ideal method for detecting the molecules with the sensitivity, specificity, and robustness needed for clinical applications. Indeed, in their Tri-Con posters, Zhou and colleagues cited a recent Clinical Chemistry interview in which five miRNA experts agreed that qPCR, at least near term, is the most likely technology to succeed in the analysis of miRNAs in the clinical lab.

"MicroRNAs are huge, and the problem is the existing assays are not quite robust enough," Zhou said. "We see a lot of potential, but we are also seeing a lot of interplatform variations and assay imprecision. This is what really drove us to optimize our platform."

Zhou said that A*Star, where he also serves as a research scientist, was an early innovator in the field of miRNA analysis, having filed for its first patents covering miRNA analysis and detection in the early 2000s, then optimizing the method and refiling for new patents around 2010, eventually yielding the mSMRT-qPCR platform.

Each mSMRT-qPCR assay comprises three miRNA-specific oligonucleotides: a stem-looped reverse transcriptase primer that efficiently captures the mature but not precursor form of target miRNA and then converts it to cDNA; and two nested qPCR primers that can specifically amplify this cDNA. SYBR Green is used for detection.

"It uses a specific microRNA RT primer, with a stem loop to capture," Zhou said, noting that this is similar to the way Life Technologies' TaqMan assays for miRNA analysis work. "But moving forward, as you hit the qPCR stage, you have a microRNA-specific forward primer and a microRNA-specific reverse primer," he noted.

"SYBR Green will make this whole reaction very fast, and having three specific primers for each microRNA … really enables the specificity and sensitivity for each miRNA," Zhou added.

MiRxes also has at its disposal a proprietary algorithm that was developed by thermodynamic statistical modeling of more than 5,000 sets of experimental data, and computes the optimal primer combinations to maximize miRNA detection sensitivity while minimizing non-specific interactions with homologous sequences or among primers.

"We spent [a year and a half] to really understand the DNA/RNA hybridization, and how you can effectively put three sets of primers on such a short miRNA [of about] 22 base pairs," Zhou said.

MiRxes said that it validates all of its assays with synthetic miRNA and biological samples, and that the assays are designed to comply with the minimum information for publication of quantitative real-time PCR experiments, or MIQE, guidelines.

Finally, the A*Star and MiRxes scientists have designed the assay technology to be agnostic in terms of qPCR chemistries and instruments.

"We tried it with more than 20 different kinds of qPCR mix; we tried it with various RT mixes; we tried it with all kinds of qPCR machines, and they all worked, with optimization," Zhou said. "Ultimately it's a qPCR assay, and it should work, using the protocol.

"When we designed it, the whole idea was to do something that people were familiar with — we're trying to enable researchers and clinicians without changing current settings," he added. "So you can take the assays, use the protocol you're familiar with, just do a routine qRT-PCR, and you get the data."

In its Tri-Con poster, MiRxes provided very early in-house data showing that mSMRT-qPCR assays have high reproducibility, having comparably measured 250 miRNAs in 30 human cancer sera in two independent laboratories one year apart.

The company also said that the assay technology enables a greater than 100-fold improvement in sensitivity over "major competitors" in detecting circulating miRNAs with a detection limit at 500 copies/ml of serum or plasma. It did not identify the competing commercial products, but the field is still relatively uncluttered, with companies such as Life Tech, Exiqon, Qiagen, and Quanta Biosciences among the notable vendors of such assays.

Development hub

MiRxes is pursuing two business strategies with its technology platform as the driver. First, it is offering the platform as a research tool to academic, diagnostics, and pharma customers that are interested in exploring various miRNAs as biomarkers for any number of applications.

"You give me any small RNA — so this is not limited to miRNA, we can also do this for siRNA, for instance — and we can very easily design robust assays," Zhou said. "For that we offer a life science research tool platform, very much similar to existing qPCR assays for miRNA, but this is just better."

In addition, MiRxes is part of a national initiative in Singapore's National Initiative on Diagnostics, a countrywide public-private technology transfer and development effort that includes A*Star, various spinoff companies like MiRxes, and clinical partners such as the National University Health System, National Cancer Centre Singapore, Singapore General Hospital, and the National University Heart Centre, Singapore.

"The technology from MirXes is a result of my organization — we fund projects, gap projects," Emril Ali, manager of biomedical sciences at Exploit Technologies, A*Star's tech-transfer organization. "The source of the technologies is research institutes, universities — basically a patent that hasn't been developed yet. We fund that development and the result is companies coming out in the early phases."

With regards to diagnostics development, this funding gap is closing, Ali said, but not fast enough, "so a level of development is then pushed downstream. This requires development [on our part] in line with how the industry does it. We have to start doing the verification and validation to industry standards, and lay the groundwork … hence the formation of the Diagnostics Development Hub. This is the entity where we will start partnering with commercial [entities]."

Toward that end, MiRxes and others in the initiative have already begun exploring the development of mSMRT-qPCR assays for the non-invasive early detection of gastric cancer and breast cancer, Zhou said.

"Gastric cancer is a huge problem in Asia, not so much the US and Europe, so we're pushing to develop a lab-based test for that, and hopefully we'll be launching that in Singapore, also Korea and China, in perhaps two years' time," Zhou said. "We are at the beginning of clinical testing. For breast cancer we are talking to partners."

But MiRxes' more immediate goal, Zhou noted, is to get the mSMRT-qPCR into the hands of potential customers so they can develop and validate their own assays and perhaps choose to strike a partnership with the development hub.

Importantly, Zhou noted, the group does not plan to draw the line at miRNA biomarkers.

"We realized that circulating miRNAs offers a lot of information that is complementary to existing biomarkers such as protein biomarkers, or even circulating DNA biomarkers," Zhou said. "We believe this is a different class of molecules, master regulators, so they do offer a different set of information. One direction we are pushing heavily is to combine these with existing biomarkers. We're not saying we will introduce this microRNA test that will replace everything. We believe that in the future personalized medicine will be a combination of different kinds and classes of biomarkers."

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