Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum

TLDR

Exosomes are abundant cell‑derived vesicles in biological fluids that carry RNA molecules, making them promising diagnostic biomarkers for precision medicine. This study compares and validates multiple exosome and exoRNA isolation methods to facilitate their clinical application and provide a methodological reference. Exosomes from cell‑culture medium and serum were isolated by ultracentrifugation, ExoQuick, or TEI, while exoRNA was extracted with TRIzol‑LS, SeraMir, TER, HLR, miRNeasy, or exoRNeasy and quantified by NanoDrop, Bioanalyzer, qPCR, and high‑throughput sequencing. Ultracentrifugation yielded the fewest particles but highest protein purity; TEI+TER achieved high small‑RNA extraction efficiency from CCM; exoRNeasy produced high‑yield, narrow‑size small RNA from serum; overall, method choice markedly affected exosome and exoRNA concentration, purity, and size distributions.

Abstract

Exosomes are cell-derived vesicles and are abundant in biological fluids; they contain RNA molecules which may serve as potential diagnostic biomarkers in 'precision medicine'. To promote the clinical application of exosomal RNA (exoRNA), many isolation methods must be compared and validated. Exosomes in cell culture medium (CCM) and serum may be isolated using ultracentrifugation (UC), ExoQuick or Total Exosome Isolation Reagent (TEI), and exoRNA may be extracted using TRIzol-LS, SeraMir, Total Exosome RNA Isolation (TER), HiPure Liquid RNA/miRNA kit (HLR), miRNeasy or exoRNeasy. ExoRNA was assessed using NanoDrop, Bioanalyzer 2100, quantitative polymerase chain reaction and high-throughput sequencing. UC showed the lowest recovery of particles, but the highest protein purity for exosome isolation. For isolation of exoRNA, we found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM. High yield and a narrow size distribution pattern of small RNA were shown in exoRNA isolated by exoRNeasy from serum. In RNA profile analysis, the small RNA constituent ratio, miRNA content and amount varied as a result of methodological differences. This study showed that different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA. Herein we discuss the advantages and disadvantages of each method and their application to different materials, therefore providing a reference according to research design.