Fig. 1: Workflow in the extracellular RNA Quality Control (exRNAQC) study.

To evaluate the 8 exRNA purification methods (upper left panel), 2 blood draws from a single individual were performed to separately apply mRNA capture or miRNA sequencing. To compare RNA purification performance, 9 performance metrics were calculated. Blood was drawn from 9 individuals to evaluate 10 blood collection tube types, including 5 classic and 5 preservation tube types (upper right panel), at 3 time intervals between blood draw and processing. Preservation tubes were processed immediately (T0) and after 24 (T24) and 72 (T72) hours and classic tubes were processed immediately (T0) and after 4 (T04) and 16 (T16) hours. Both mRNA capture and miRNA sequencing were performed, and the data was analyzed using 5 performance metrics. Based on the number of miRNAs and mRNAs detected and replicate variability metrics, a dedicated selection of precise and sensitive exRNA purification methods and blood collection tubes was further evaluated in exRNAQC phase 2. For both mRNA capture and miRNA sequencing in phase 2, 5 individuals were sampled to test 3 blood collection tubes and 4 RNA purification methods. Interactions between RNA purification methods, blood collection tubes and processing time intervals were assessed by 6 performance metrics. MAP=MagNA Pure method, MAX=Maxwell method, MIR=miRNeasy method, MIRA=miRNeasy Advanced method, MIRV=mirVana method, MIRVE=mirVana method with purification protocol for small RNA enrichment, NOR=Norgen method, NUC=NucleoSpin method, QIA=QIAamp method. Designed with Freepik (free license) and Servier Medical Art (CC BY 4.0).