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Urinary extracellular vesicles : Assessment of pre-analytical variables and development of a quality control with focus on transcriptomic biomarker research

Barreiro, Karina ; Dwivedi, Om Prakash ; Valkonen, Sami ; Groop, Per Henrik ; Tuomi, Tiinamaija LU orcid ; Holthofer, Harry ; Rannikko, Antti ; Yliperttula, Marjo ; Siljander, Pia and Laitinen, Saara , et al. (2021) In Journal of Extracellular Vesicles 10(12).
Abstract

Urinary extracellular vesicles (uEV) are a topical source of non-invasive biomarkers for health and diseases of the urogenital system. However, several challenges have become evident in the standardization of uEV pipelines from collection of urine to biomarker analysis. Here, we studied the effect of pre-analytical variables and developed means of quality control for uEV isolates to be used in transcriptomic biomarker research. We included urine samples from healthy controls and individuals with type 1 or type 2 diabetes and normo-, micro- or macroalbuminuria and isolated uEV by ultracentrifugation. We studied the effect of storage temperature (-20°C vs. -80°C), time (up to 4 years) and storage format (urine or isolated uEV) on quality... (More)

Urinary extracellular vesicles (uEV) are a topical source of non-invasive biomarkers for health and diseases of the urogenital system. However, several challenges have become evident in the standardization of uEV pipelines from collection of urine to biomarker analysis. Here, we studied the effect of pre-analytical variables and developed means of quality control for uEV isolates to be used in transcriptomic biomarker research. We included urine samples from healthy controls and individuals with type 1 or type 2 diabetes and normo-, micro- or macroalbuminuria and isolated uEV by ultracentrifugation. We studied the effect of storage temperature (-20°C vs. -80°C), time (up to 4 years) and storage format (urine or isolated uEV) on quality of uEV by nanoparticle tracking analysis, electron microscopy, Western blotting and qPCR. Urinary EV RNA was compared in terms of quantity, quality, and by mRNA or miRNA sequencing. To study the stability of miRNA levels in samples isolated by different methods, we created and tested a list of miRNAs commonly enriched in uEV isolates. uEV and their transcriptome were preserved in urine or as isolated uEV even after long-term storage at -80°C. However, storage at -20°C degraded particularly the GC-rich part of the transcriptome and EV protein markers. Transcriptome was preserved in RNA samples extracted with and without DNAse, but read distributions still showed some differences in e.g. intergenic and intronic reads. MiRNAs commonly enriched in uEV isolates were stable and concordant between different EV isolation methods. Analysis of never frozen uEV helped to identify surface characteristics of particles by EM. In addition to uEV, qPCR assays demonstrated that uEV isolates commonly contained polyoma viruses. Based on our results, we present recommendations how to store and handle uEV isolates for transcriptomics studies that may help to expedite standardization of the EV biomarker field.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
biomarkers, DNAse, microRNA, mRNA, storage temperature, storage time, transcriptomics, urinary exosomes, urinary extracellular vesicles, virus
in
Journal of Extracellular Vesicles
volume
10
issue
12
article number
e12158
publisher
Co-Action Publishing
external identifiers
  • pmid:34651466
  • scopus:85117387469
ISSN
2001-3078
DOI
10.1002/jev2.12158
language
English
LU publication?
yes
additional info
Publisher Copyright: © 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles
id
202de3e9-3090-46ef-9d84-0f1f4072cfbd
date added to LUP
2021-11-22 11:15:40
date last changed
2024-09-22 05:53:49
@article{202de3e9-3090-46ef-9d84-0f1f4072cfbd,
  abstract     = {{<p>Urinary extracellular vesicles (uEV) are a topical source of non-invasive biomarkers for health and diseases of the urogenital system. However, several challenges have become evident in the standardization of uEV pipelines from collection of urine to biomarker analysis. Here, we studied the effect of pre-analytical variables and developed means of quality control for uEV isolates to be used in transcriptomic biomarker research. We included urine samples from healthy controls and individuals with type 1 or type 2 diabetes and normo-, micro- or macroalbuminuria and isolated uEV by ultracentrifugation. We studied the effect of storage temperature (-20°C vs. -80°C), time (up to 4 years) and storage format (urine or isolated uEV) on quality of uEV by nanoparticle tracking analysis, electron microscopy, Western blotting and qPCR. Urinary EV RNA was compared in terms of quantity, quality, and by mRNA or miRNA sequencing. To study the stability of miRNA levels in samples isolated by different methods, we created and tested a list of miRNAs commonly enriched in uEV isolates. uEV and their transcriptome were preserved in urine or as isolated uEV even after long-term storage at -80°C. However, storage at -20°C degraded particularly the GC-rich part of the transcriptome and EV protein markers. Transcriptome was preserved in RNA samples extracted with and without DNAse, but read distributions still showed some differences in e.g. intergenic and intronic reads. MiRNAs commonly enriched in uEV isolates were stable and concordant between different EV isolation methods. Analysis of never frozen uEV helped to identify surface characteristics of particles by EM. In addition to uEV, qPCR assays demonstrated that uEV isolates commonly contained polyoma viruses. Based on our results, we present recommendations how to store and handle uEV isolates for transcriptomics studies that may help to expedite standardization of the EV biomarker field.</p>}},
  author       = {{Barreiro, Karina and Dwivedi, Om Prakash and Valkonen, Sami and Groop, Per Henrik and Tuomi, Tiinamaija and Holthofer, Harry and Rannikko, Antti and Yliperttula, Marjo and Siljander, Pia and Laitinen, Saara and Serkkola, Elina and af Hällström, Taija and Forsblom, Carol and Groop, Leif and Puhka, Maija}},
  issn         = {{2001-3078}},
  keywords     = {{biomarkers; DNAse; microRNA; mRNA; storage temperature; storage time; transcriptomics; urinary exosomes; urinary extracellular vesicles; virus}},
  language     = {{eng}},
  number       = {{12}},
  publisher    = {{Co-Action Publishing}},
  series       = {{Journal of Extracellular Vesicles}},
  title        = {{Urinary extracellular vesicles : Assessment of pre-analytical variables and development of a quality control with focus on transcriptomic biomarker research}},
  url          = {{http://dx.doi.org/10.1002/jev2.12158}},
  doi          = {{10.1002/jev2.12158}},
  volume       = {{10}},
  year         = {{2021}},
}