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.
Detailed results are available as a preprint.
Anneleen Decock, Olivier De Wever, Celine Everaert, Hetty Hilde Helsmoortel, An Hendrix, Pieter Mestdagh, Annelien Morlion, Jo Vandesompele, & Ruben Van Paemel
Francisco Avila Cobos, Anneleen Decock, Celine Everaert, Hetty Hilde Helsmoortel, Jan Koster, Annelien Morlion, Franco Poma-Soto, Ruben Van Paemel, & Jasper Verwilt
Jasper Anckaert, Francisco Avila Cobos, Jilke De Wilde, Celine Everaert, Carolina Fierro, Pieter Mestdagh, Annelien Morlion, Franco Poma-Soto, Jo Vandesompele, Ruben Van Paemel, &Jasper Verwilt
Anneleen Decock, Pieter Mestdagh, & Jo Vandesompele
Anneleen Decock, Philippe Decruyenaere, Jill Deleu, Jilke De Wilde, Bert Dhondt, Hetty Hilde Helsmoortel, Eva Hulstaert, Nele Nijs, Justine Nuytens, Annouck Philippron, Kathleen Schoofs, Eveline Vanden Eynde, Ruben Van Paemel, Kimberly Verniers, Jasper Verwilt & Nurten Yigit
Francisco Avila Cobos, Anneleen Decock, Bert Dhondt, Thibaut D’huyvetter, Celine Everaert, Carolina Fierro, Hetty Hilde Helsmoortel, Pieter Mestdagh, Annelien Morlion, Nele Nijs, Annouck Philippron, Thomas Piofczyk, Olivier Thas, Jo Vandesompele & Ruben Van Paemel
Philippe Decruyenaere, Jilke De Wilde, Bert Dhondt, Eva Hulstaert, Jan Koster, Scott Kuersten, Tim R. Mercer, Annouck Philippron, Gary P. Schroth & Ruben Van Paemel
Jasper Anckaert, Francisco Avila Cobos, Celine Everaert, Annelien Morlion, Olivier Thas, Ruben Van Paemel & Jasper Verwilt
Katleen De Preter, Pieter Mestdagh, Jo Vandesompele & Tom Van Maerken
Francisco Avila Cobos, Anneleen Decock, Jilke De Wilde, Celine Everaert, Jan Koster, Annelien Morlion, Franco Poma-Soto, Kathleen Schoofs, Ruben Van Paemel & Jasper Verwilt
Writing - original draft
Francisco Avila Cobos, Anneleen Decock, Philippe Decruyenaere, Jill Deleu, Jilke De Wilde, Celine Everaert, Annelien Morlion, Kathleen Schoofs, Ruben Van Paemel & Jasper Verwilt
Writing - review & editing
Anneleen Decock, Celine Everaert, Pieter Mestdagh, Annelien Morlion & Jo Vandesompele