A pipeline for the development and analysis of extracellular vesicle-based transcriptomic biomarkers in molecular diagnostics

Extracellular vesicles are shed by every cell type and can be found in any biofluid. They contain different molecules that can be utilized as biomarkers, including several RNA species which they protect from degradation. Here, researchers at the Technical University of Munich present a pipeline for the development and analysis of extracellular vesicle-associated transcriptomic biomarkers that their group has successfully applied multiple times. The researchers highlight the key steps of the pipeline and give particular emphasis to the necessary quality control checkpoints, which are linked to numerous available guidelines that should be considered along the workflow. This pipeline starts with patient recruitment and continues with blood sampling and processing. The purification and characterization of extracellular vesicles is explained in detail, as well as the isolation and quality control of extracellular vesicle-associated RNA. They point out the possible pitfalls during library preparation and RNA sequencing and present multiple bioinformatic tools to pinpoint biomarker signature candidates from the sequencing data. Finally, considerations and pitfalls during the validation of the biomarker signature using RT-qPCR will be elaborated.

Graphical representation of the EV-based transcriptomic biomarker identification pipeline from RNA extraction to bioinformatics, with focus on the pitfalls connected with EV-RNA analysis and helpful guidelines and tools available for each step

Grätz C, Schuster M, Brandes F, Meidert AS, Kirchner B, Reithmair M, Schelling G, Pfaffl MW. (2024) A pipeline for the development and analysis of extracellular vesicle-based transcriptomic biomarkers in molecular diagnostics. Mol Aspects Med 97:101269. [article]

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