Mapping bacterial extracellular vesicle research: insights, best practices and knowledge gaps.

Bacterial extracellular vesicles (BEVs) enable communication between bacteria and their natural habitats, including multicellular organisms such as humans. Consequently, the study of BEVs has rapidly gained attention with recent research raising the prospect of developing BEVs as biomarkers and treatments to manage (mal)functioning of natural habitats. Although diverse technologies are available, the composition of their source, their heterogeneity in biophysical and biochemical features, and their multifaceted cargo composition challenges the analysis of BEVs. To map current practices in BEV research, we analyzed 845 publications released in 2015-2021, reporting 3338 BEV-related experiments. The extracted data are accessible via the publicly available EV-TRACK knowledgebase ( https://evtrack.org/ ). We identify the need for transparent reporting, delineate knowledge gaps, outline available best practices and define areas in need of guidance to ensure advances in BEV research and accelerate BEV applications.

Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data.

Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications.