Quantification of EV-associated miRNA in liquid biopsies for biomarker signature development

Extracellular vesicle (EV)-associated microRNAs (miRNAs) are promising minimally invasive biomarkers, as EV encapsulation protects miRNAs from degradation while preserving disease- and cell- type-specific expression patterns across diverse human body fluids. This review summarizes the current understanding of miRNA and EV biogenesis, including the mechanisms governing EV cargo packaging. We further discuss the broad range of EV sources used in liquid biopsy and molecular diagnostics, including blood, urine, saliva, milk, respiratory fluids, and cerebrospinal fluid, highlighting both the diagnostic utility and key pre-analytical challenges associated with EV-miRNA analysis. State-of-the-art EV isolation approaches, including differential ultracentrifugation, size-exclusion chromatography, affinity-based methods, microfluidic platforms, flow cytometry and nanoparticle-based analyses are not isolation methods and are therefore excluded from this comparison. In addition, workflows for EV-associated RNA extraction and quantification, including RNA sequencing and reverse transcription-quantitative polymerase chain reaction-based methodologies, are critically evaluated. Emphasis is placed on challenges related to data quality assessment, normalization strategies, low-input sample analysis, and high sample heterogeneity. This review also summarizes current standardization initiatives, including MISEV, MIQE, EV-TRACK, and EV Task Force biofluid guidelines, emphasizing the importance of rigorous reporting standards, harmonized pre-analytical workflows, and multiparametric normalization strategies for reproducible detection and accurate quantification. Finally, we discuss emerging best practices and unresolved challenges in multivariate modeling, miRNA isoform-based analyses, and in silico validation of the target recognition elements and regulatory pathways. An integrated, guideline-based workflow from liquid biopsy collection to clinically actionable EV-miRNA signatures is proposed to facilitate translation of this approach into routine molecular diagnostics.