Although extracellular RNAs are promising emerging liquid biopsy analytes, lack of benchmarked exRNA quantification procedures (that are robust and sensitive) hampers discovery and clinical implementation of exRNA biomarkers. The importance of having standardized procedures for sample collection, processing and profiling is underscored by the fact that our own experience and published liquid biopsy studies have demonstrated that many pre-analytical factors substantially influence exRNA quantification results and that the impact of these factors on the analytical result varies markedly depending on the profiling technology (e.g. massively parallel sequencing versus PCR) 1 2. While such small-scale studies have pointed at the impact of individual preanalytical factors, no study has systematically and comprehensively addressed the many pre-analytical variables affecting downstream sequencing of blood-derived exRNAs. Therefore, we set up the Extracellular RNA Quality Control (exRNAQC) study, a collaborative initiative between our lab and different industrial partners (Biogazelle, Illumina, Roche, Promega and Qiagen) in which the influence of selected pre-analytical variables on both small RNA and mRNA sequencing of exRNA is systematically being assessed.
The exRNAQC study comprises three phases, whereby we evaluate the type of blood collection tube (n=10, including 5 preservation plasma tubes), the time between blood draw and plasma preparation (n=3) and RNA purification methods using the supplier specified minimum and maximum plasma input volumes (n=15). The impact of these pre-analytical factors is assessed by unbiased transcriptome profiling of small and messenger RNAs from healthy donors’ plasma, using TruSeq Small RNA sequencing and TruSeq RNA Exome sequencing (Illumina), respectively. Importantly, we make use of 191 synthetic RNA spike-in molecules, enabling to control the entire workflow. The first phase of the study, in which blood collection tubes and RNA extraction kits are evaluated separately, is completed. Results show huge performance differences among tubes and kits. When comparing blood collection tubes, serum mRNA seems very similar to EDTA plasma mRNA, but serum-derived small RNAs are markedly different in biotype composition compared to their plasma counterparts. Furthermore, several plasma tubes with preservation reagents do not stabilize RNA very well, as is reflected by increasing RNA concentrations and number of detected genes over time. Also, their reproducibility is generally compromised. In addition, we demonstrate large differences in RNA purification kit performance in terms of reproducibility, sensitivity and observed transcriptome complexity. Based on these data, a selection of 3 tubes and 4 extraction kits was put forward for a more in-depth analysis in the second phase of exRNAQC, assessing interactions between different pre-analytical factors. In the third phase, the impact of freeze/thaw cycles and varying plasma and RNA input levels will be studied, and a fully nested variant component analysis will be performed. This is paramount ground work for any future RNA-based liquid biopsy-guided precision oncology study.
CONCEPTUALIZATION: Ruben Van Paemel, Pieter Mestdagh, Olivier De Wever, Jo Vandesompele, Anneleen Decock, Hetty Hilde Helsmoortel, Annelien Morlion, Celine Everaert, An Hendrix.
DATA CURATION: Ruben Van Paemel, Francisco Avila Cobos, Anneleen Decock, Hetty Hilde Helsmoortel, Annelien Morlion, Celine Everaert.
FORMAL ANALYSIS: Jo Vandesompele, Ruben Van Paemel, Pieter Mestdagh, Annelien Morlion, Jilke De Wilde, Francisco Avila Cobos, Celine Everaert, Carolina Fierro.
FUNDING ACQUISITION: Anneleen Decock, Jo Vandesompele, Pieter Mestdagh.
INVESTIGATION: Eveline Vanden Eynde, Nele Nijs, Jilke De Wilde, Bert Dhondt, Eva Hulstaert, Annouck Philippron, Ruben Van Paemel, Jill Deleu, Justine Nuytens, Kimberly Verniers, Kathleen Schoofs, Hetty Hilde Helsmoortel, Anneleen Decock, Nurten Yigit.
METHODOLOGY: Annelien Morlion, Pieter Mestdagh, Jo Vandesompele, Anneleen Decock, Celine Everaert, Carolina Fierro, Ruben Van Paemel, Annouck Philippron, Bert Dhondt, Nele Nijs, Francisco Avila Cobos, Hetty Hilde Helsmoortel.
PROJECT ADMINISTRATION: Anneleen Decock.
RESOURCES: Bert Dhondt, Ruben Van Paemel, Jilke De Wilde, Gary Schroth, Scott Kuersten, Annouck Philippron, Eva Hulstaert. SOFTWARE: Francisco Avila Cobos, Ruben Van Paemel, Annelien Morlion, Celine Everaert, Jasper Anckaert.
SUPERVISION: Pieter Mestdagh, Jo Vandesompele.
Wong RKY et al. (2019). A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma. BMC Genomics 20(1):446. ↩︎
Laurent LC et al. (2015). Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium. J Extracell Vesicles 4:26533. ↩︎