Blood collection tube and RNA purification method recommendations for extracellular RNA transcriptome profiling

Blood-based extracellular RNA (cell-free RNA; exRNA) biomarkers require validated sample collection, processing, and quantification procedures. No study to date has systematically tested pre-analytical variables affecting transcriptome-wide exRNA analysis. By evaluating their impact on deep transcriptome profiling of microRNAs and mRNAs in blood plasma or serum, we compared ten blood collection tubes, three blood processing time intervals, and eight RNA purification methods. In addition, we assessed interactions among a selected pre-analytical variable set, resulting in 456 extracellular transcriptomes. Blood preservation tubes failed to stabilize exRNA and RNA purification methods differed significantly in performance, causing variations in concentration, detected gene numbers, replicability and observed transcriptome complexity. Critical interactions between tubes, purification methods and time intervals were identified. We provide 11 analytical performance metrics for exRNA quantification methods and put forward recommendations for both users and manufacturers of RNA purification methods and blood collection tubes, collectively, essential groundwork for exRNA-based precision medicine applications.

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Publication Details

Journal
Nature Communications
Published
2025-05-15
DOI
https://doi.org/10.1038/s41467-025-58607-7
Citations
13
Primary Topic
Molecular Biology Techniques and Applications
Type
article
Field-Weighted Citation Impact
10.48

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article

Blood collection tube and RNA purification method recommendations for extracellular RNA transcriptome profiling

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13 citations
Nature Communications
Molecular Biology Techniques and Applications
10.48
article

Blood collection tube and RNA purification method recommendations for extracellular RNA transcriptome profiling

Bert Dhondt, Francisco Avila Cobos, Eva Hulstaert, Annouck Philippron, Celine Everaert, Philippe Decruyenaere, Gary P. Schroth, Thomas Piofczyk, Kimberly Verniers, Carolina Bustos Fierro, Jasper Anckaert, Jilke De Wilde, Olivier De Wever, Thibaut D’huyvetter, Nele Nijs, Justine Nuytens, Jill Deleu, Scott Kuersten, Ruben Van Paemel, Franco Poma-Soto, Pieter Mestdagh, Tom Van Maerken, Nurten Yigit, Anneleen Decock, An Hendrix, Hetty Helsmoortel, Kathleen Schoofs, Jasper Verwilt, Eveline Vanden Eynde, Jo Vandesompele, Annelien Morlion, Tim R. Mercer, Jan Köster, Olivier Thas, Katleen De Preter
article en
13 citations

Abstract

Blood-based extracellular RNA (cell-free RNA; exRNA) biomarkers require validated sample collection, processing, and quantification procedures. No study to date has systematically tested pre-analytical variables affecting transcriptome-wide exRNA analysis. By evaluating their impact on deep transcriptome profiling of microRNAs and mRNAs in blood plasma or serum, we compared ten blood collection tubes, three blood processing time intervals, and eight RNA purification methods. In addition, we assessed interactions among a selected pre-analytical variable set, resulting in 456 extracellular transcriptomes. Blood preservation tubes failed to stabilize exRNA and RNA purification methods differed significantly in performance, causing variations in concentration, detected gene numbers, replicability and observed transcriptome complexity. Critical interactions between tubes, purification methods and time intervals were identified. We provide 11 analytical performance metrics for exRNA quantification methods and put forward recommendations for both users and manufacturers of RNA purification methods and blood collection tubes, collectively, essential groundwork for exRNA-based precision medicine applications.

Nature CommunicationsVol. 16(1)
HOGENT University of Applied Sciences and Arts (BE), Australian Bureau of Statistics (AU), Garvan Institute of Medical Research (AU), The University of Queensland (AU), Illumina (United States) (US), University of Wollongong (AU), AZ Groeninge (BE), Ghent University Hospital (BE), Ghent University (BE), Cancer Research Institute Ghent (BE), Amsterdam University Medical Centers (NL), Hasselt University (BE)
European Commission, Fonds Wetenschappelijk Onderzoek, Universiteit Gent, Kom op tegen Kanker, Vlaamse regering, Fondation contre le Cancer, Bijzonder Onderzoeksfonds UGent
Zero hunger
Openalex Percentile: Top 1%
Molecular Biology Techniques and Applications
10.48
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