Extracellular vesicles, RNA sequencing, and bioinformatic analyses: Challenges, solutions, and recommendations

Extracellular vesicles and particles (EVPs) are heterogeneous entities secreted by cells that carry functional molecular cargo, including small RNAs, across systemic circulation. These particles are increasingly recognized as vital messengers for intercellular communication and as promising candidates for liquid biopsy diagnostics and targeted therapeutic delivery. However, the field currently lacks a gold standard for transcriptomic analysis, leading to significant variability in reported findings across studies.

The primary obstacles to robust EVP transcriptomics include the low concentration of RNA recovered from biofluids, the high heterogeneity of particle populations, and the use of sequencing protocols originally designed for cellular RNA. Standard quality control metrics, such as the RNA Integrity Number (RIN), are often inappropriate for the short, fragmented RNA species typically found in EVPs. Furthermore, bioinformatic pipelines often struggle with high rates of multimapping reads and a lack of standardized databases, which complicates the accurate identification and quantification of EV-associated RNA cargo.

To address these gaps, the authors advocate for a more rigorous approach to EVP research. Key recommendations include:

• Integrating ultrafiltration with specific molecular weight cut-offs (MWCO) to improve sample purity and concentration.
• Utilizing capillary electrophoresis (e.g., Pico and small RNA kits) rather than traditional RIN scores to assess RNA quality.
• Incorporating RNA spike-in controls to normalize sequencing data and evaluate recovery efficiency.
• Adopting specialized bioinformatics pipelines that account for the unique nature of short-length and unannotated RNA species, rather than relying on standard cellular RNA workflows.