Absolute Quantification of microRNA Copies in Exosomes Using Real‐Time PCR

Exosomes are small extracellular vesicles (50 to 150 nm in size) that carry different kind of biomolecules in their lumen and facilitate intercellular communication. Serum contains a large number of exosomes (∼1012/ml of serum) that contain relatively more small RNAs than large RNAs. Quantifying the absolute number of specific miRNA molecules in exosomes is critical for understanding their functional relevance and developing them for diagnostic and therapeutic applications. This article describes an exosome copy number assay that combines nanoparticle tracking analysis (NTA) to quantify vesicle concentration with absolute quantification of miRNA using a standard curve-based qPCR approach. The workflow includes optimized procedures for vesicle isolation, particle measurement, RNA extraction, and absolute quantification, enabling calculation of miRNA copy number per exosome. The method can be used to generate reproducible and accurate results across diverse biological samples. By offering precise molecular quantification, these protocols provide a practical framework to advance exosome biology and support the development of exosome-based biomarkers and therapies. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Isolation of exosomes from human serum by ultracentrifugation Support Protocol: Exogenous spike-in controls and troubleshooting Basic Protocol 2: NTA for exosome enumeration Basic Protocol 3: Extraction of RNA from purified exosomes from serum Basic Protocol 4: Reverse transcription (cDNA synthesis and preamplification) and real-time PCR setup Basic Protocol 5: Copy number assay (standard curve preparation) Basic Protocol 6: Calculation of miRNA copy number per vesicle (absolute quantification by qPCR and NTA normalization).