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Chinese Scientists Publish New Isothermal miRNA Detection Method with Point-of-Care Potential

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NEW YORK (GenomeWeb) — A Chinese team has published the details of an isothermal nucleic acid amplification method for highly sensitive microRNA detection.

According to the investigators from the State Key Laboratory of Chemo/Biosensing and Chemometrics at Hunan University, the new technique has comparable amplification efficiency to more established nucleic acid amplification methods, but is less susceptible to non-specific amplification.

The method can also be employed in a closed-tube environment in a single step and in real time, and can potentially be used for miRNA expression profiling and related theranostic applications, the researchers said.

MicroRNA has the potential to be a non-invasive biomarker of disease, enabling molecular diagnostics and theranostics, with miRNA signatures having already been implicated as proxies for brain injury, cardiovascular disease, and cancer. MiRNA are also proposed targets of therapeutics. However, miRNAs are usually only present in trace amounts, and their tiny size of around 20 nucleotides and general instability makes detecting them via standard methods tricky.

In the past few years, a number of different methods have been developed to detect and discriminate miRNAs. These include RT-PCR followed by qPCR, droplet digital PCR, miRNA sequencing, microarrays, and electrochemical methods involving carbon nanotubes, to name a few. Many of these techniques require complex equipment, limiting them to use in lab environments.

The emerging field of isothermal miRNA detection intends to bring miRNA detection to the point of care. However, the field is so new — some two dozen methods have been published since 2012, and only four prior to that — there has yet to be any comprehensive review of the various technologies.

Although all the methods in the literature describe ways to detect miRNA using a single temperature, they vary in their molecular methodologies. Some use enzymatic tricks, like nickases, while others use hairpins or rolling circle amplification, for example.

The newly developed technique, described in a letter last week to Analytical Chemistry, is a one-step method with a fluorescent readout. In an email with PCR Insider, Jian-Hui Jiang, corresponding author on the study, asserted that "aberrant expression of miRNAs is closely implicated in various diseases, including cancers, diabetes, neurological disorders, cardiovascular and autoimmune diseases."

Jiang also confirmed the lack of a literature review in this new field. By his own assessment, published isothermal miRNA detection methods now include amplification by rolling circle, exponential strand-displacement, modified invader, hairpin-mediated quadratic enzymatic amplification, and duplex-specific nuclease signal amplification.

Add to this his group's method, called exponential amplification reaction, or ERA. It is novel in that it essentially uses the miRNA as a primer. If a miRNA of interest — in the case of the current study, miR-21 — is present in a sample, it can bind and cause amplification of another piece of DNA, resulting in a fluorescent readout.

Jiang explained that the method is also based on the DNA base excision repair process.

In the method, inserted uracil lesion bases in a DNA duplex are excised by uracil-DNA glycosylase and endonuclease IV, creating a single nucleotide gap, Jiang said. DNA polymerase then fills in the gap. "However, after the extension by polymerase, the lesion bases are replaced by the abnormal nucleotides ... resulting in a cyclic repair for signal amplification and detection," Jiang explained.

Importantly, Jiang noted that compared with more commonly employed nickase-based nucleic acid amplification technologies, "the ERA reaction provided comparable amplification efficiency with improved resistance to non-specific amplification, independent of primers and templates." The method also showed "excellent specificity, with the capability of discriminating single-base mismatch," he said.

When used for miRNA detection, the ERA strategy could also be implemented in a single-step, closed-tube, and real-time detection format, Jiang said, enabling convenient operation and resistance to contaminants. "The strategy was shown to give a detection limit as low as 0.1 fM for miRNA detection across a wide dynamic range up to 1 nM."

The method was used to measure miR-21 levels in a handful of cancer cell lines, according to the study. Jiang said he believes it has the potential to be used for miRNA detection in real, complex samples, although work is ongoing on this front.

The developing field of isothermal miRNA detection, however, must also keep pace with the changes in the miRNA-as-biomarker worldview. Diagnostics and theranostics developers especially must also keep an eye on sobering voices in the field.

A recent report, for example, suggested choice of technology may introduce biases in miRNA detection, with different sequencing methods producing different results from the same samples.

And, although miRNA has been touted as a biomarker for everything from cancer to transplant rejection, some oncology researchers are now urging the field to curb its enthusiasm.

In a review published online in March in Molecular Oncology's special issue on biomarker discovery, researchers pored over miRNA studies from the past five years. The result: "a total of 154 diagnostic, prognostic, and/or predictive circulating miRNA signatures in the field of cancer research alone."

Yet this group found little overlap between different studies of purported biomarker signatures for a given tumor type, and also uncovered variability in sample procurement and data analysis, according to the review. "The lack of adherence to MIQE guidelines of almost all studies makes a retrospective analysis of the results, as well as true duplication thereof in an independent setting difficult, if not impossible," the authors concluded.

Thus, whether there will be a need for a bedside isothermal miRNA detection in the future remains to be seen.

"Our method might hold the potential for miRNA expression profiling, but it still needs much work," Jiang said. He added that his group does not have any commercial partners as yet, and he was unaware of any commercialization efforts for other isothermal miRNA detection techniques.

It is worth noting that in the same issue of Analytical Chemistry, researchers from another Chinese institution — the College of Chemistry and Material Science at Shandong Agricultural University — described another isothermal miRNA detection strategy. This method employs T7 exonuclease and cyclic enzymatic amplification, and uses DNA probes assembled onto a surface made of gold.

These researchers demonstrated that their method also had a high level of specificity, discriminating a single-base mismatched microRNA sequence, and achieved a low detection limit of 0.17 fM. Taking more of an agricultural bent — although one with possible human health implications — the group described how its assay could be used to detect avian leukemia based on the decreased expression level of miR-21.

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