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.
Competing Interest Statement
Carolina Fierro and Nele Nijs are employees, Thomas Piofczyk is a former employee, Pieter Mestdagh is a consultant, and Jo Vandesompele a co-founder of Biogazelle, a clinical CRO providing human biofluid extracellular RNA sequencing. Gary P. Schroth and Scott Kuersten are employees of Illumina, providing library preparation and sequencing reagents. Promega, Qiagen and Roche sponsored blood collection tubes and/or RNA purification kits. Funders did not influence data analysis, interpretation and manuscript writing.
Footnotes
↵* Within the CRediT groups, authors are in alphabetical order. Annelien Morlion and Ruben Van Paemel took the lead in the analysis of the RNA purification kit and blood collection tube study, respectively.
Conflict of interest Carolina Fierro and Nele Nijs are employees, Thomas Piofczyk is a former employee, Pieter Mestdagh is a consultant, and Jo Vandesompele a co-founder of Biogazelle, a clinical CRO providing human biofluid extracellular RNA sequencing. Gary P. Schroth and Scott Kuersten are employees of Illumina, providing library preparation and sequencing reagents. Promega, Qiagen and Roche sponsored blood collection tubes and/or RNA purification kits. Funders did not influence data analysis, interpretation and manuscript writing.
Abbreviations ACD-A: BD Vacutainer Glass ACD Solution A tube; ALC: area left of the curve; Biomatrica: LBgard Blood Tube; BRISQ: Biospecimen Reporting for Improved Study Quality; bp: base pair; CCF: QIAamp ccfDNA/RNA Kit; CIRC: Plasma/Serum Circulating and Exosomal RNA Purification Kit/Slurry Format; citrate: Vacuette Tube 9 ml 9NC Coagulation sodium citrate 3.2%; DNA Streck: Cell-Free DNA BCT; EDTA: BD Vacutainer Plastic K2EDTA tube; EDTA separator: Vacuette Tube 8 ml K2E K2EDTA Separator; ERCC: Extracellular RNA Communication Consortium; exRNA: extracellular RNA; FC: fold change; gDNA: genomic DNA; LP: Library Prep Control; MAP: MagNA Pure 24 Total NA Isolation Kit in combination with the MagNA Pure instrument; MAX: Maxwell RSC miRNA Plasma and Exosome Kit in combination with the Maxwell RSC Instrument; MIR: miRNeasy Serum/Plasma Kit; MIRA: miRNeasy Serum/Plasma Advanced Kit; miRNA: microRNA; MIRV: mirVana PARIS Kit with purification protocol for total RNA; MIRVE: mirVana PARIS Kit with purification protocol for RNA enriched for small RNAs; mRNA: messenger RNA; NUC: NucleoSpin miRNA Plasma Kit; PAXgene: PAXgene Blood ccfDNA Tube; RA3: RNA 3’ adapter; RA5: RNA 5’ adapter; RC: RNA extraction Control; RNA Streck: Cell-Free RNA BCT; Roche: Cell-Free DNA Collection Tube; serum: BD Vacutainer SST II Advance Tube; SOP: standard operating procedure;