Precise RNA Quantification by Counting Individual RNA Molecules Using High-Sensitivity Capillary Flow Cytometry.

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.
• 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.