Multiplexed and accurate quantification strategy for miRNA based on specific terminal-mediated PCR with equivalent...

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• MicroRNAs (miRNAs) are crucial biomarkers for disease diagnosis and prognosis.
• Accurate, multiplexed quantification of miRNAs is challenging due to their complex functions and lack of a universal reference gene.

Purpose of the Study:

• To develop a novel, sensitive, and specific multiplexed miRNA detection method.
• To establish a single-tube assay for simultaneous quantification of multiple miRNAs.
• To evaluate the performance and clinical applicability of the new method.

Main Methods:

• Developed Specific Terminal-Mediated miRNA PCR (STEM-Mi-PCR).
• Utilized target-specific capture primers for linear reverse transcription.
• Employed universal primers for exponential amplification in a multiplex assay.

Main Results:

• Achieved high sensitivity (100 aM) and equivalent amplification efficiency (95.67 ± 8.58%) in a 4-plex assay.
• Demonstrated high specificity with no cross-reactivity between detected miRNAs.
• Quantified miRNAs in patient tissues across pM to fM concentrations, showing practical applicability.
• Exhibited single-nucleotide mutation discrimination capabilities with minimal nonspecific signals.

Conclusions:

• STEM-Mi-PCR offers a sensitive, specific, and efficient method for multiplexed miRNA profiling.
• The assay is suitable for quantifying miRNAs in clinical samples.
• This technique provides a promising platform for future miRNA-based diagnostics and research.