Check your RNA quality with excellent speed and accuracy on Lunatic

Significance of the Topic

High RNA purity is essential for reliable downstream applications such as quantitative RT-PCR, RNA sequencing and lipid nanoparticle formulation. Contaminating DNA, proteins or residual extraction reagents can generate background signal, inhibit enzymatic steps and compromise quantitative accuracy. Rapid, accurate assessment of RNA quality and concentration is therefore a cornerstone of genomic research and therapeutic development.

Objectives and Study Overview

This application note evaluates the performance of the Lunatic microfluidic UV/Vis system equipped with Unmix algorithms to:

• Quantify RNA concentration across a wide dynamic range using only 2 µL samples.
• Differentiate RNA absorbance from co-absorbing contaminants including DNA, proteins and residual extraction buffers.
• Assess precision, accuracy and linearity in the presence of defined DNA and protein spikes.

Methodology and Instrumentation

Purified calf liver RNA, calf thymus DNA and bovine serum albumin (BSA) spiked samples were prepared at varying ratios and total nucleic acid concentrations. Measurements employed High Lunatic plates with 96 microfluidic circuits, reading up to 96 samples in 10 minutes using 2 µL per well. UV/Vis spectra were recorded from 230 to 350 nm. Unmix deconvolution algorithms corrected for background turbidity and separated RNA, DNA and protein contributions without dyes or standard curves. Data outliers were removed by interquartile range filtering and processed in RStudio.

Main Results and Discussion

Precision: Six replicates of 1,000 ng/µL RNA yielded overlaying spectra with low standard deviation and coefficient of variation, demonstrating high reproducibility.

DNA Contamination: Samples spiked with 5–40% DNA maintained linear recovery of RNA concentration (R² = 0.9925). The Unmix application accurately decreased reported RNA values proportional to DNA content while simultaneously reporting impurity levels.

Concentration Range: RNA/DNA mixtures diluted from 1,000 to 62.5 ng/µL consistently preserved the expected RNA fraction (70% ±2%), confirming robust performance across a broad dynamic range.

Protein Contamination: BSA spikes at 10% and 50% (v/v) were correctly identified by Unmix, and the measured RNA concentration adjusted proportionally to dilution by protein, highlighting routine detection of protein impurities.

Benefits and Practical Applications

Lunatic offers rapid, high-throughput assessment of RNA quality and concentration without consumables such as dyes or standards. By distinguishing co-absorbing contaminants, the system reduces false positives in downstream assays, streamlines QC workflows in research and industry, and conserves valuable sample material.

Future Trends and Potential Applications

Advancements may include integration with automated sample handling and laboratory information management systems, expanded Unmix libraries for additional contaminants, adaptation for small RNAs and chemically modified transcripts, and deployment in large-scale production QC for RNA therapeutics.

Conclusion

The Lunatic microfluidic UV/Vis system equipped with Unmix algorithms delivers rapid, precise and accurate RNA quantification in the presence of DNA, proteins and extraction reagents. Its minimal sample requirement and high throughput make it a valuable tool for genomics research, diagnostic assay development and therapeutic manufacturing.

Reference

Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159 (1987).