Blood collection tube and RNA purification method recommendations for extracellular RNA transcriptome profiling
Jasper Anckaert, 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, Carolina Bustos Fierro, Hetty Helsmoortel, An Hendrix, Eva Hulstaert, Jan Köster, Scott Kuersten, Tim R. Mercer, Pieter Mestdagh, Annelien Morlion, Nele Nijs, Justine Nuytens, Annouck Philippron, Thomas Piofczyk, Franco 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
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. Here the authors provide a comprehensive evaluation of important pre-analytical variables affecting extracellular RNA quantification and analysis in the exRNAQC study, examining eight RNA purification methods, ten blood collection tubes, and three time intervals for blood processing.