Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation

Area of Science:

• Biochemistry and Molecular Biology
• Genomics and Bioinformatics
• Biomarker Discovery

Background:

• Lack of knowledge on extracellular RNA (exRNA) isolation method performance hinders reproducibility in the exRNA field.
• Inconsistent results across studies impede progress in exRNA research and biomarker development.

Purpose of the Study:

• To systematically compare the performance of 10 different exRNA isolation methods.
• To evaluate the impact of these methods on small RNA sequencing profile complexity and reproducibility.
• To develop a tool to assist researchers in selecting the optimal exRNA isolation method.

Main Methods:

• Compared 10 exRNA isolation methods across 5 distinct biofluids.
• Assessed method efficiency by analyzing proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures.
• Developed an interactive web-based application (miRDaR) for comparative analysis and method prioritization.

Main Results:

• Significant differences were observed in the complexity and reproducibility of small RNA sequencing profiles generated by different exRNA isolation methods.
• Method efficiency varied in accessing different exRNA carrier subclasses (EVs, RNPs, HDLs).
• The miRDaR application provides comparative statistics and ranks methods based on complexity, expression level, and reproducibility.

Conclusions:

• The choice of exRNA isolation method significantly impacts downstream small RNA sequencing results.
• The miRDaR tool can guide researchers in selecting appropriate methods, thereby improving experimental reproducibility.
• Standardized method selection will accelerate progress in exRNA biomarker discovery and validation.