Extracellular vesicles (EVs) have significant potential as therapeutic agents and as sources of diagnostic, predictive, and prognostic nucleic acid biomarkers. However, variability in EV workflows and inadequate standardization of downstream analysis pose major obstacles to reproducibility. The MISEV (Minimal Information for Studies of Extracellular Vesicles) guidelines provide essential domain-specific recommendations for EV isolation, characterization, analysis, nomenclature, and reporting, but deliberately refrain from prescribing methods for the molecular quantification of EV cargo. Among the analytical platforms used in EV studies, quantitative reverse transcription PCR (RT-qPCR) is the most critical method for validating and quantifying EV-associated RNA. The recently revised MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 2.0 guidelines offer a detailed foundation for ensuring analytical validity in RT-qPCR-based quantitative applications. The proposed model of harmonizing general and domain-specific guidelines provides a scalable blueprint for improving reproducibility across complex biomarker development workflows in molecular diagnostics.
Keywords: MIQE; MISEV; RT-qPCR; analytical validity; biomarker research; extracellular vesicles; reproducibility; standardization.
© The Author(s) 2025.