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Circulating EVs carry functional small RNAs and other molecular footprints from their cell of origin, and thus have evident applications in liquid biopsy, therapeutics, and intercellular communication. Yet, the complete transcriptomic landscape of EVs is poorly characterized due to critical limitations including variable protocols used for EV\u2010RNA extraction, quality control, cDNA library preparation, sequencing technologies, and bioinformatic analyses. Consequently, there is a gap in knowledge and the need for a standardized approach in delineating EV\u2010RNAs. Here, we address these gaps by describing the following points by (1) focusing on the large canopy of the EVs and particles (EVPs), which includes, but not limited to \u2013 exosomes and other large and small EVs, lipoproteins, exomeres\/supermeres, mitochondrial\u2010derived vesicles, RNA binding proteins, and cell\u2010free DNA\/RNA\/proteins; (2) examining the potential functional roles and biogenesis of EVPs; (3) discussing various transcriptomic methods and technologies used in uncovering the cargoes of EVPs; (4) presenting a comprehensive list of RNA subtypes reported in EVPs; (5) describing different EV\u2010RNA databases and resources specific to EV\u2010RNA species; (6) reviewing established bioinformatics pipelines and novel strategies for reproducible EV transcriptomics analyses; (7) emphasizing the significant need for a gold standard approach in identifying EV\u2010RNAs across studies; (8) and finally, we highlight current challenges, discuss possible solutions, and present recommendations for robust and reproducible analyses of EVP\u2010associated small RNAs. Overall, we seek to provide clarity on the transcriptomics landscape, sequencing technologies, and bioinformatic analyses of EVP\u2010RNAs. Detailed portrayal of the current state of EVP transcriptomics will lead to a better understanding of how the RNA cargo of EVPs can be used in modern and targeted diagnostics and therapeutics. For the inclusion of different particles discussed in this article, we use the terms large\/small EVs, non\u2010vesicular extracellular particles (NVEPs), EPs and EVPs as defined in MISEV guidelines by the International Society of Extracellular Vesicles (ISEV).<\/jats:p>","DOI":"10.1002\/jev2.70005","type":"journal-article","created":{"date-parts":[[2024,12,3]],"date-time":"2024-12-03T09:59:29Z","timestamp":1733219969000},"source":"Crossref","is-referenced-by-count":133,"title":["Extracellular vesicles, RNA sequencing, and bioinformatic analyses: Challenges, solutions, and recommendations"],"prefix":"10.1002","volume":"13","author":[{"given":"Rebecca T.","family":"Miceli","sequence":"first","affiliation":[{"name":"Department of Pathology, Molecular and Cell\u2010Based Medicine Icahn School of Medicine at Mount Sinai  New York New York 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