Quantification of total nucleic acids from unknown sources (RNA eq)

Quantification of Total Nucleic Acids from Unknown Sources (RNA eq) – Summary

Importance of the Topic

Reliable measurement of total nucleic acid content is critical for molecular biology workflows including cloning, sequencing, and expression studies. The ability to accurately quantify nucleic acids from diverse sources (bacterial, yeast, plant, plasmid) ensures consistent sample quality and reproducibility in downstream applications.

Goals and Overview

This application note demonstrates the use of the Nucleic Acids (RNA equivalent) Unmix application on Lunatic systems to deconvolute UV/Vis absorption spectra. By isolating the spectral signature of nucleic acids, the method provides accurate RNA-equivalent concentration measurements without distinguishing DNA from RNA.

Methodology

The Unmix algorithm analyzes full UV/Vis spectra to separate contributions from:

• Nucleic acids: quantified via the A260 peak multiplied by an RNA concentration factor (40 ng/µL per A260 unit).
• Co-absorbing impurities: reported in OD260 values, with thresholds for phenol, salts, detergents, and proteins triggering detailed reporting.
• Background turbidity: measured at OD260 and subtracted to yield a clean content spectrum, with thresholds for particles and pigments.

Quality of fit is assessed by the relative root square error (RRSE), with values above 2.5% flagged to indicate high turbidity, unknown compounds, or low concentration.

Instrumentation

The method is implemented on two platforms:

• Lunatic UV/Vis analysis system with full reporting options (HTML, XML, TXT, CSV, XLSX, PDF).
• Little Lunatic system with fixed report templates and on-screen calculations.

Main Results and Discussion

Using pure water blanks, the Unmix app provides:

• Quantitative RNA-equivalent concentration in ng/µL.
• Impurity profiles with concentration or OD values when thresholds are exceeded.
• Background profiles indicating turbidity and pigment interference.
• Quality metrics visualized as spectral overlays and RRSE percentage, ensuring data reliability.

Benefits and Practical Applications

This label-free, rapid quantification requires minimal sample input and works across varied sample matrices. It supports quality control in nucleic acid extractions, enzymatic assays, and preparative workflows.

Future Trends and Possibilities

Advancements may include expanded spectral libraries for contaminant identification, integration with automated liquid handling, and coupling with fluorescence or mass spectrometry detectors to enhance specificity and sensitivity.

Conclusion

The Nucleic Acids (RNA eq) Unmix application provides a robust and user-friendly approach for total nucleic acid quantification. Its spectral deconvolution strategy ensures accurate measurements even in complex sample backgrounds, streamlining quality control in molecular biology.