MicroRNA Technology Proves Useful for Liver Toxicity Detection

A single probe method has been developed for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases.

The sensitive detection of specific sequences of nucleic acids (NA) has become an indispensable tool in the diagnosis and treatment of diseases. The field of molecular diagnostics, where detection of specific sequences allows diagnosis of cancer, infectious diseases, and hereditary disease has emerged from these technologies.

An international team of scientists collaborating with the Quanterix Corporation, Lexington, MA, USA) collected blood samples from patients with drug-induced liver injury with the first blood sample collected within 24 hours of last acetaminophen (APAP) ingestion. A total of four adults (23 to 42 years old) were recruited to this study and for both healthy volunteers and patients with liver injury, blood samples were centrifuged immediately at 11,000 × g for 15 minutes at 4 °C, after which serum was separated into aliquots and frozen at −80 °C.

Ribonucleic acid (RNA) was isolated from serum using the miRNeasy Serum/Plasma kit. Polymerase chain reaction (PCR) was performed using a Qiagen commercial kit. An abasic peptide nucleic acid (PNA) probe containing a reactive amine instead of a nucleotide at a specific position in the sequence for detecting microRNA (miRNA) was conjugated to superparamagnetic beads. These beads were incubated with a sample containing miRNA, a biotinylated reactive nucleobase, containing an aldehyde group that was complementary to the missing base in the probe sequence, and a reducing agent. This assay was used to measure microRNA-122 (miR-122), an established biomarker of liver toxicity. To determine of concentration of miR-122 the team used using the Quanterix Simoa HD-1 Analyzer.

The high specificity (>3 × 107-fold) in the Simoa assay was achieved from the SMARTbase technology using only a single probe rather than multiple probes and primers used in PCR. The use of a single probe greatly simplified the assay designed while maintaining the ability to discriminate target sequences with single base specificity. The data indicate that the sensitivity and specificity of the Simoa assay was sufficient to measure miR-122 in all patients, and showed a clear distinction between healthy controls and those with clinical liver toxicity after drug overdose, as was the case for PCR.

Hugh A. Ilyine, the Chief Executive Officer of DestiNA Genomics, one of the companies collaborating in the study, said, “It’s an exciting breakthrough in RNA detection to learn that microRNA serve as valuable clinical biomarkers. Using a PCR-free approach that delivers a simpler, faster preparation and analysis time, but with higher accuracy, means easy microRNA detection from serum and plasma is finally possible and opens up the development of microRNA as valuable clinical biomarkers.” The study was published on July 5, 2017, in the journal Public Library of Science ONE.

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