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Summary

This summary is machine-generated.

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This study introduces a novel method for precise ribonucleic acid (RNA) quantification by directly counting individual RNA molecules, eliminating the need for reverse transcription and calibration for accurate results.

Area of Science:

Molecular Biology
Biochemistry
Analytical Chemistry

Background:

Accurate quantification of ribonucleic acid (RNA) is crucial for molecular biology research.
Current methods like reverse transcription quantitative polymerase chain reaction (RT-qPCR) can be affected by variable yields during the reverse transcription step.
Ultraviolet (UV) spectrophotometry lacks sensitivity for low-level RNA detection.

Purpose of the Study:

To develop a precise RNA quantification method that bypasses the reverse transcription step.
To enable sequence-specific counting of individual RNA molecules for accurate concentration determination.
To establish a potentially standard reference method for RNA quantification.

Main Methods:

Sequence-specific tagging of target RNA molecules with multiple fluorescent oligonucleotide probes.

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High-sensitivity capillary-based flow cytometry for exhaustive, one-by-one counting of tagged RNA molecules.

Optimization of probe number, concentration, and hybridization conditions using MS2 viral RNA as a model.

Main Results:

MS2 viral RNA was quantified with high precision (2.0% relative standard deviation) using approximately 70 oligonucleotide probes.
The method demonstrated comparability with droplet digital PCR and UV spectrophotometry, indicating minimal bias.
The technique successfully quantified RNA at levels significantly lower than UV spectrophotometry and avoided RT-qPCR variability.

Conclusions:

The developed method offers a calibration-free, sequence-specific approach for precise RNA quantification.
It overcomes limitations of existing methods, including RT variability and sensitivity issues.
This direct RNA counting technique holds potential as a valuable and accurate reference standard for RNA measurement.