Quantification of mRNA Expression Using Single-Molecule Nanopore Sensing.

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Real-time reverse transcription polymerase chain reaction (RT-qPCR) is standard for RNA quantification but can be inaccurate with rare transcripts or contaminants.
• Existing single-molecule methods often rely on fluorescence and probes, introducing noise and limiting multiplexing capabilities.

Purpose of the Study:

• To introduce reverse transcription quantitative nanopore sensing (RT-qNP) as a novel, amplification-free RNA quantification method.
• To demonstrate RT-qNP's accuracy in quantifying gene expression and viral RNA in complex biological samples.

Main Methods:

• RT-qNP involves direct synthesis and single-molecule detection of gene-specific complementary DNAs (cDNAs) without purification or amplification.
• The method was applied to quantify metastasis-associated genes (MACC1, S100A4) in human cell lines and SARS-CoV-2 RNA.
• An internal reference gene was used for accurate quantification, eliminating the need for calibration curves.

Main Results:

• RT-qNP accurately quantified relative gene expression of MACC1 and S100A4 in cell lines, even when RT-qPCR yielded uncertain results.
• The method successfully quantified SARS-CoV-2 RNA against a human reference gene.
• RT-qNP demonstrated minimal sample loss and suitability for direct nanopore sensing of complicated biological samples.

Conclusions:

• RT-qNP provides sensitive, label-free, single-molecule RNA quantification without amplification.
• This method overcomes limitations of current RNA quantification techniques, offering accurate detection of low RNA levels and viral infections.
• RT-qNP is a versatile tool for early disease detection and precise quantification in complex biological samples.