Automated enrichment and co-extraction pipeline for efficient analysis of urinary extracellular vesicle protein and RNA

Extracellular vesicles (EVs) cargoes have great potential as promising biomarkers for disease diagnosis. However, traditional methods for EV enrichment and cargo extraction are inefficient, limiting the accessibility of valuable biological information. Here, we present a bead-based pipeline, designed to efficiently enrich EVs from urine while simultaneously extracting both EV protein and RNA. Compared to traditional methods involving ultracentrifugation (UC) for EV isolation, acetone precipita-tion for protein extraction, and TRIzol for RNA extraction, the new pipeline achieved over a 5-fold improvement in EV protein and RNA yield. Additionally, it reduced the whole duration from approximately 2 days to just 1 h. When applied to the non-invasive diagnosis of prostate cancer (PCa), the pipeline demonstrated excellent diagnostic performance, with a panel of EV protein and RNA markers achieving an AUC of 0.82. This efficient and streamlined pipeline provides a robust tool for com-prehensive EV cargo analysis and holds great potential for advancing non-invasive diagnostics in clinical settings.