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Optimized procedure for high-throughput transcriptome profiling of small extracellular vesicles isolated from low volume serum samples

P. Vychytilova-Faltejskova, S. Vilmanova, L. Pifkova, T. Catela Ivković, M. Mᶏdrzyk, R. Jugas, T. Machackova, J. Kotoucek, M. Sachlova, L. Bohovicova, T. Stanek, J. Halamkova, I. Kiss, O. Slaby

. 2024 ; 62 (1) : 157-167. [pub] 20230731

Jazyk angličtina Země Německo

Typ dokumentu časopisecké články

Perzistentní odkaz   https://www.medvik.cz/link/bmc24000074

OBJECTIVES: Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome. METHODS: Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays. RESULTS: Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype. CONCLUSIONS: We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.

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$a Vilmanova, Sara $u Centre for Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic
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$a Pifkova, Lucie $u Centre for Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic
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$a Catela Ivković, Tina $u Centre for Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic
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$a Mᶏdrzyk, Marie $u Centre for Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic
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$a Machackova, Tana $u Centre for Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic
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$a Kotoucek, Jan $u Department of Pharmacology and Toxicology, Veterinary Research Institute, Brno, Czech Republic
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$a Sachlova, Milana $u Department of Gastroenterology and Digestive Endoscopy, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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$a Bohovicova, Lucia $u Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Faculty of Medicine, Brno, Czech Republic
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$a Stanek, Teodor $u Department of Surgical Oncology, Masaryk Memorial Cancer Institute, Faculty of Medicine, Brno, Czech Republic
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$a Halamkova, Jana $u Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Faculty of Medicine, Brno, Czech Republic
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$a Kiss, Igor $u Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Faculty of Medicine, Brno, Czech Republic
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$a Slaby, Ondrej $u Centre for Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic $u Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
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