Isothermal circular strand displacement-based assay for microRNA detection in liquid biopsy

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Extracellular microRNAs (miRNAs) are key biomarkers for disease diagnosis and prognosis via liquid biopsy.
• Challenges in miRNA detection include short sequences, low concentrations, and biological fluid interferences.
• Isothermal circular strand displacement polymerization offers a viable amplification and detection method.

Purpose of the Study:

• To develop an innovative assay for direct microRNA detection in biological fluids.
• To optimize a microfluidic device for analyzing multiple complex samples.
• To validate the assay for detecting specific microRNAs linked to disease.

Main Methods:

• Utilized hairpin probe-assisted isothermal amplification for miRNA detection.
• Employed a microfluidic device for analyzing small sample volumes (1 µL).
• Incorporated a high-yield mutant polymerase and locked nucleic acid nucleosides in primers.

Main Results:

• Achieved a limit of detection (LOD) of 34 fmol L⁻¹ for miR-127-5p.
• Successfully quantified circulating miR-127-5p in synovial fluid, a biomarker for osteoarthritis.
• Demonstrated detection of miRNAs at concentrations as low as 4.3 ± 0.5 pmol L⁻¹.

Conclusions:

• The developed assay enables sensitive and direct detection of extracellular miRNAs in complex biological matrices.
• This method holds potential for early disease diagnosis and prognosis through liquid biopsy.
• The assay's efficiency in complex fluids paves the way for routine clinical applications.