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

Vychytilova-Faltejskova, Petra ; Vilmanova, Sara ; Pifkova, Lucie ; Catela Ivković, Tina LU ; Mᶏdrzyk, Marie ; Jugas, Robin ; Machackova, Tana ; Kotoucek, Jan ; Sachlova, Milana and Bohovicova, Lucia , et al. (2024) In Clinical Chemistry and Laboratory Medicine 62(1). p.157-167
Abstract

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:... (More)

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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publishing date
type
Contribution to journal
publication status
published
keywords
Humans, Gene Expression Profiling/methods, Extracellular Vesicles/genetics, RNA, Chromatography, Gel
in
Clinical Chemistry and Laboratory Medicine
volume
62
issue
1
pages
157 - 167
publisher
De Gruyter
external identifiers
  • scopus:85167417058
  • pmid:37505924
ISSN
1434-6621
DOI
10.1515/cclm-2023-0610
language
English
LU publication?
no
additional info
© 2023 the author(s), published by De Gruyter, Berlin/Boston.
id
f512515a-e8e2-4878-b1da-1e1702800e32
date added to LUP
2025-02-11 21:37:45
date last changed
2025-06-04 12:57:55
@article{f512515a-e8e2-4878-b1da-1e1702800e32,
  abstract     = {{<p>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.</p><p>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.</p><p>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.</p><p>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.</p>}},
  author       = {{Vychytilova-Faltejskova, Petra and Vilmanova, Sara and Pifkova, Lucie and Catela Ivković, Tina and Mᶏdrzyk, Marie and Jugas, Robin and Machackova, Tana and Kotoucek, Jan and Sachlova, Milana and Bohovicova, Lucia and Stanek, Teodor and Halamkova, Jana and Kiss, Igor and Slaby, Ondrej}},
  issn         = {{1434-6621}},
  keywords     = {{Humans; Gene Expression Profiling/methods; Extracellular Vesicles/genetics; RNA; Chromatography, Gel}},
  language     = {{eng}},
  month        = {{01}},
  number       = {{1}},
  pages        = {{157--167}},
  publisher    = {{De Gruyter}},
  series       = {{Clinical Chemistry and Laboratory Medicine}},
  title        = {{Optimized procedure for high-throughput transcriptome profiling of small extracellular vesicles isolated from low volume serum samples}},
  url          = {{http://dx.doi.org/10.1515/cclm-2023-0610}},
  doi          = {{10.1515/cclm-2023-0610}},
  volume       = {{62}},
  year         = {{2024}},
}