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Review
. 2024 Dec;13(12):e70005.
doi: 10.1002/jev2.70005.

Extracellular vesicles, RNA sequencing, and bioinformatic analyses: Challenges, solutions, and recommendations

Rebecca T Miceli  1 , Tzu-Yi Chen  1 , Yohei Nose  2   3 , Swapnil Tichkule  4 , Briana Brown  1 , John F Fullard  4   5   6   7 , Marilyn D Saulsbury  8 , Simon O Heyliger  8 , Sacha Gnjatic  2   3   9 , Natasha Kyprianou  1   3   10 , Carlos Cordon-Cardo  1 , Susmita Sahoo  9   11 , Emanuela Taioli  12   13 , Panos Roussos  4   5   6   7   14   15 , Gustavo Stolovitzky  5   16 , Edgar Gonzalez-Kozlova  2   3 , Navneet Dogra  1   5   17   18
Affiliations
Review

Extracellular vesicles, RNA sequencing, and bioinformatic analyses: Challenges, solutions, and recommendations

Rebecca T Miceli et al. J Extracell Vesicles. 2024 Dec.

Abstract

Extracellular vesicles (EVs) are heterogeneous entities secreted by cells into their microenvironment and systemic circulation. 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-RNA 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-RNAs. 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 - exosomes and other large and small EVs, lipoproteins, exomeres/supermeres, mitochondrial-derived vesicles, RNA binding proteins, and cell-free 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-RNA databases and resources specific to EV-RNA 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-RNAs across studies; (8) and finally, we highlight current challenges, discuss possible solutions, and present recommendations for robust and reproducible analyses of EVP-associated small RNAs. Overall, we seek to provide clarity on the transcriptomics landscape, sequencing technologies, and bioinformatic analyses of EVP-RNAs. 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-vesicular extracellular particles (NVEPs), EPs and EVPs as defined in MISEV guidelines by the International Society of Extracellular Vesicles (ISEV).

Keywords: EVs; bioinformatics; extracellular vesicles; long‐read sequencing; short‐read sequencing; small RNA; transcriptomics.

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Conflict of interest statement

S.G. reports past consultancy or advisory roles for Taiho Pharmaceuticals; research funding from Regeneron Pharmaceuticals, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Genentech, EMD Serono, Pfizer and Takeda, unrelated to the current work. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. N.D. and G.S. are awarded a patent entitled ‘Exosome vessels for delivery of molecular cargo’, US Patent 11,766,484.

Figures

FIGURE 1
FIGURE 1
A summary of experimental and computation tools used for EV and RNA isolation and transcriptomic analysis of small RNAs from extracellular vesicles and particles. Current obstacles within each section are highlighted in red and discussed in Section 8. DG, density gradient; nanoDLD, nanofluidic deterministic lateral displacement; SEC, size exclusion chromatography; UC, ultracentrifugation.
FIGURE 2
FIGURE 2
The biogenesis of various EVP subtypes, including exosomes, exomeres, supermeres and mitochondrial‐derived vesicles. Multivesicular bodies (MVB) are specialized ‘late’ endosomes derived from the plasma membrane that are sorted for lysosomal degradation or recycling via the Golgi apparatus. Intraluminal vesicles (ILVs) are formed when the membrane of the MVBs folds inward towards the lumen and leads to the budding of vesicles into the MVB lumen. ILVs may be transported to the plasma membranes and released extracellularly as exosomes. Mitochondria‐derived vesicles (MDVs) are formed under normal physiological or stress‐related conditions. During oxidative stress, the mitochondria may shed oxidized membrane proteins or damaged proteins through the budding off the damaged segments of the mitochondrial membrane for eventual degradation. Exomeres and supermeres are small (<50 nm) non‐membranous particles containing lipids, nucleic acids and proteins that are co‐isolated with small EVs from unknown biogenesis pathways.
FIGURE 3
FIGURE 3
Overview of the RNA landscape of EVPs. Most commonly studied small EVP subtypes ranging from a diameter of ∼1 nm to >200 nm (top), and the most common types of RNA found within EVPs ranging from miRNA, piRNA, tRNA, snRNA, YRNA, circRNA, snoRNA, lncRNAs, mRNA and rRNA (bottom).
FIGURE 4
FIGURE 4
EV functionality: challenges and opportunities. Current challenges and potential opportunities with EV‐RNA functionality.
See this image and copyright information in PMC

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