Quality Assessment of Total RNA Using MultiNA Automated Analysis

Significance of the Topic

The integrity of total RNA is a critical parameter in diverse molecular biology applications, including northern blotting, cDNA library construction, PCR, and next-generation sequencing. Traditional denaturing gel electrophoresis requires extensive manual handling, is time-consuming, and the qualitative assessment of band patterns can be subjective. Automated microchip electrophoresis platforms offer rapid, reproducible, and quantitative evaluation of RNA quality, enhancing throughput and consistency in research and diagnostic settings.

Objectives and Study Overview

This study evaluated the MultiNA™ microchip electrophoresis system for automated quality assessment of total RNA extracted from rat kidney tissue. The primary goal was to monitor changes in the 28S/18S rRNA ratio as a function of storage time at room temperature (0, 1, and 2 weeks) and to demonstrate the system’s capacity for objective, numerical reporting of RNA integrity.

Methodology and Instrumentation

• Sample Preparation: Rat kidney total RNA stock (1 µg/µL) diluted to 50 ng/µL in TE buffer.
• Thermal Denaturation: Each RNA sample and RNA 6000 Ladder solution heated at 65 °C for 5 minutes followed by rapid cooling at 4 °C for 5 minutes.
• Electrophoresis Conditions: Samples mixed with the supplied marker in a 1:1 ratio and separated on the MultiNA system using SYBR Green II dye for fluorescence detection.

Instrumentation Used

• MCE-202 MultiNA™ Microchip Electrophoresis System
• RNA kit (Shimadzu)
• SYBR Green II fluorescent dye (Thermo)
• RNA 6000 Ladder marker (Thermo)

Main Results and Discussion

• Automated Peak Identification: The MultiNA system consistently identified 18S and 28S rRNA peaks alongside a low molecular weight marker in all samples.
• Quantitative Integrity Metrics: Calculated 28S/18S ratios were 2.70 at week 0, 1.84 at week 1, and 1.63 at week 2, reflecting progressive RNA degradation over time.
• Objective Quality Evaluation: Numerical outputs enabled clear discrimination between high-quality RNA (ratio near 2:1) and degraded samples, demonstrating the system’s suitability for rigorous quality control.

Benefits and Practical Applications

• Fully Automated Workflow: Minimizes manual handling and reduces variability between runs.
• Objective Numerical Reporting: Enables standardized quality thresholds and easier data comparison across experiments.
• High Throughput Capability: Supports large sample sets, making it ideal for core facilities and high-volume laboratories.

Future Trends and Potential Applications

• Integration into Sequencing Pipelines: Real-time RNA quality checks prior to library preparation could improve data reliability in next-generation sequencing.
• Expanded Assay Portfolio: Development of protocols for small RNA species and partially degraded samples to broaden analytical scope.
• Advanced Data Analytics: Implementation of machine learning algorithms to predict sample suitability and optimize storage and handling protocols.

Conclusion

The MultiNA microchip electrophoresis system streamlines total RNA quality assessment by combining rapid, automated separation with objective numerical metrics. Its robust performance in detecting RNA degradation over storage time and its high throughput capabilities make it a valuable tool for research and diagnostic laboratories seeking consistent and efficient RNA integrity evaluation.

References

1. Shimadzu Corporation. Application News: Quality Assessment of Total RNA Using MultiNA. First Edition: May 2023.