Substantional performance differences among RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control study - important considerations for liquid biopsies

Cancer biomarker studies require procedures that provide accurate and precise test results with high analytical sensitivity. Consequently, the growing use of extracellular RNA from human biofluids as clinically relevant biomarker requires the implementation of benchmarked methods for sample collection, processing and profiling. While several small-scale studies have pointed at the impact of individual pre-analytical factors, no comprehensive study has addressed the many pre-analytical variables affecting downstream sequencing of blood-derived exRNAs. In the Extracellular RNA Quality Control study, we have systematically evaluated the type of blood collection tube (n=10, including 5 so-called preservation tubes), the time between blood draw and plasma preparation (n=3), different plasma types (n=3, i.e. platelet-free, -poor, and -rich plasma), 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 deep transcriptome profiling of all small and messenger RNAs from healthy donors’ plasma, using TruSeq Small RNA sequencing and TruSeq RNA Exome sequencing, respectively. All experiments are conducted in triplicate (for a total of 270 transcriptomes) using 191 synthetic RNA spike-in molecules as processing controls over a relevant dynamic range. 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. Amongst others, we note a 50-fold difference in mRNA yield and a 5-fold difference in the number of detected mRNAs. We summarized the results in 12 performance parameters that enable an informed selection of the most optimal sample processing workflow. In conclusion, using a systematic approach, we put forward robust quality control metrics for exRNA quantification methods with validated SOPs for sample collection, processing and profiling. Our results are crucially important for all future RNA-based liquid biopsy-guided precision oncology applications.