Quantitative Distribution of Intact RNA in EVs and Non-EVs Fractions from Healthy Human Plasma
GAN Xingyi, LIN Xinyi*, XIAO Xuhuang, GUO Jiahui, CUI Yizhi, WANG Tong*
This study aims to utilize the optimized dSEC (dichotomic size-exclusion chromatography) technique to achieve efficient, high-purity separation of plasma EVs (extracellular vesicles) and non-EVs components. Plasma EVs and non-EVs components were isolated using the dSEC method. The isolated fractions were systematically characterized according to MISEV2023 guidelines, utilizing NTA (nanoparticle tracking analysis), Western blot, and TEM (transmission electron microscopy). Transcriptome analysis was conducted via NGS (next-generation sequencing). RNA quantification was defined by raw counts>10, and high-integrity RNAs were screened using a sequencing coverage threshold>95%. The composition, integrity, and distribution differences of high-integrity RNAs between the two fractions were systematically compared. The dSEC method efficiently isolated high-purity plasma EVs, which exhibited a significantly higher particle-to-protein ratio than the non-EVs fraction and met MISEV2023 standards. A total of 31 284 and 39 224 stably quantified RNAs were identified in the EVs and non-EVs fractions, respectively, with 29 108 RNAs common to both. LncRNAs (long non-coding RNAs) and protein-coding RNAs were the predominant RNA biotypes in both fractions; however, approximately 99% of these long RNAs existed in a fragmented form. No significant differences were observed between the two fractions regarding the abundance distribution of highintegrity RNAs across major biotypes, including coding RNAs, lncRNAs, pseudogenes, and miRNAs. Notably, fulllength RNY4 was the only RNA molecule found to be stably expressed with high abundance in the EVs fraction (mean TPM was 2 019.55) while being completely undetected in the non-EVs fraction. This study systematically reveals the distribution patterns of high-integrity RNA in healthy human plasma EVs versus non-EVs components, clarifying their RNA composition and integrity profiles. Crucially, this study identified a specific full-length expression signature of RNY4 in plasma EVs. These findings provide novel experimental data for understanding RNA distribution across different plasma carriers and lay a foundation for standardizing EVs RNA research and liquid biopsy detection systems.
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