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Performance evaluation of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

Jasper Anckaert (UGent) , Francisco Avila Cobos (UGent) , Anneleen Decock (UGent) , Philippe Decruyenaere (UGent) , Jill Deleu (UGent) , Katleen De Preter (UGent) , Olivier De Wever (UGent) , Jilke De Wilde (UGent) , Bert Dhondt (UGent) , Thibaut D'huyvetter (UGent) , et al.
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Abstract
The use of blood-based extracellular RNA (exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated 10 blood collection tubes, 3 time points between blood draw and downstream processing, and 8 RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics is assessed by deep transcriptome profiling of both small and messenger RNA from healthy donors’ plasma or serum. Experiments are conducted in triplicate (for a total of 276 transcriptomes) using 189 synthetic spike-in RNAs as processing controls. When comparing blood tubes, so-called blood preservation tubes do not stabilize RNA very well, as is reflected by increasing RNA concentration and number of detected genes over time, and by compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow for your own experiments. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated standard operating procedures (SOPs) for processing, representing paramount groundwork for future exRNA-based precision medicine applications.
Keywords
extracellular RNA (exRNA), cell-free RNA (cfRNA), liquid biopsies, plasma, serum, pre-analytical variables, pre-analytics

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MLA
Anckaert, Jasper, et al. “Performance Evaluation of RNA Purification Kits and Blood Collection Tubes in the Extracellular RNA Quality Control (ExRNAQC) Study.” BioRXiv, 2022, doi:10.1101/2021.05.11.442610.
APA
Anckaert, J., Avila Cobos, F., Decock, A., Decruyenaere, P., Deleu, J., De Preter, K., … Consortium, E. (2022). Performance evaluation of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study. https://doi.org/10.1101/2021.05.11.442610
Chicago author-date
Anckaert, Jasper, Francisco Avila Cobos, Anneleen Decock, Philippe Decruyenaere, Jill Deleu, Katleen De Preter, Olivier De Wever, et al. 2022. “Performance Evaluation of RNA Purification Kits and Blood Collection Tubes in the Extracellular RNA Quality Control (ExRNAQC) Study.” BioRXiv. https://doi.org/10.1101/2021.05.11.442610.
Chicago author-date (all authors)
Anckaert, Jasper, Francisco Avila Cobos, Anneleen Decock, Philippe Decruyenaere, Jill Deleu, Katleen De Preter, Olivier De Wever, Jilke De Wilde, Bert Dhondt, Thibaut D’huyvetter, Celine Everaert, Ana Carolina Elisa Fierro Gutierrez, Hetty Helsmoortel, An Hendrix, Eva Hulstaert, Jan Koster, Scott Kuersten, Tim R. Mercer, Pieter Mestdagh, Annelien Morlion, Nele Nijs, Justine Nuytens, Annouck Philippron, Thomas Piofczyk, Franco Alexander Poma Soto, Kathleen Schoofs, Gary P. Schroth, Olivier Thas, Eveline Vanden Eynde, Jo Vandesompele, Tom Van Maerken, Ruben Van Paemel, Kimberly Verniers, Jasper Verwilt, Nurten Yigit, and ExRNAQC Consortium. 2022. “Performance Evaluation of RNA Purification Kits and Blood Collection Tubes in the Extracellular RNA Quality Control (ExRNAQC) Study.” BioRXiv. doi:10.1101/2021.05.11.442610.
Vancouver
1.
Anckaert J, Avila Cobos F, Decock A, Decruyenaere P, Deleu J, De Preter K, et al. Performance evaluation of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study. BioRXiv. 2022.
IEEE
[1]
J. Anckaert et al., “Performance evaluation of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study,” BioRXiv. 2022.
@misc{01GS7J0KB635CXHRRMVCHFA0AP,
  abstract     = {{The use of blood-based extracellular RNA (exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated 10 blood collection tubes, 3 time points between blood draw and downstream processing, and 8 RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics is assessed by deep transcriptome profiling of both small and messenger RNA from healthy donors’ plasma or serum. Experiments are conducted in triplicate (for a total of 276 transcriptomes) using 189 synthetic spike-in RNAs as processing controls. When comparing blood tubes, so-called blood preservation tubes do not stabilize RNA very well, as is reflected by increasing RNA concentration and number of detected genes over time, and by compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow for your own experiments. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated standard operating procedures (SOPs) for processing, representing paramount groundwork for future exRNA-based precision medicine applications.}},
  author       = {{Anckaert, Jasper and Avila Cobos, Francisco and Decock, Anneleen and Decruyenaere, Philippe and Deleu, Jill and De Preter, Katleen and De Wever, Olivier and De Wilde, Jilke and Dhondt, Bert and D'huyvetter, Thibaut and Everaert, Celine and Fierro Gutierrez, Ana Carolina Elisa and Helsmoortel, Hetty and Hendrix, An and Hulstaert, Eva and Koster, Jan and Kuersten, Scott and Mercer, Tim R. and Mestdagh, Pieter and Morlion, Annelien and Nijs, Nele and Nuytens, Justine and Philippron, Annouck and Piofczyk, Thomas and Poma Soto, Franco Alexander and Schoofs, Kathleen and Schroth, Gary P. and Thas, Olivier and Vanden Eynde, Eveline and Vandesompele, Jo and Van Maerken, Tom and Van Paemel, Ruben and Verniers, Kimberly and Verwilt, Jasper and Yigit, Nurten and Consortium, ExRNAQC}},
  keywords     = {{extracellular RNA (exRNA),cell-free RNA (cfRNA),liquid biopsies,plasma,serum,pre-analytical variables,pre-analytics}},
  language     = {{eng}},
  series       = {{BioRXiv}},
  title        = {{Performance evaluation of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study}},
  url          = {{http://doi.org/10.1101/2021.05.11.442610}},
  year         = {{2022}},
}

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