Journal Article

Systematic study of exRNA isolation reveals presence of distinct exRNA carriers

Lapidus, Jodi, Breakefield, Xandra O, Das, Saumya, Gandhi, Roopali, Laurent, Clara, Zhang, Xuan, Shah, Ravi, Yan, Irene, De Hoff, Peter, Simonson, Bridget, Laurent, Lucie, To, Cuong, Filant, Justyna, Srinivasan, Srimeenakshi, Sood, Anil, Nejad, Parham, Patel, Tushar, Paul, Anu, Danielson, Kirsty, Balaj, Leonora, Laurent, Louise C, Cheah, Pike See

2018

Overview

Background: Extracellular RNAs (exRNAs) have lately spawned a lot of interest as potential biomarkers, mediators of intercellular communication and therapeutic agents. ExRNA study is challenging due to the impact of biological variables on exRNA levels and technical concerns, such as low abundance and biases in methods used for isolation and analysis. Here, we systematically investigated a variety of isolation methods on standardized biofluids across multiple sites. Methods: Total and carrier enriched exRNA was isolated from five biofluids. Precipitation, membrane filtration, ultracentrifugation or affinity purification was used for exRNA carrier enrichment. exRNA was then extracted from total biofluid and the exRNA carrier enriched fractions. Small RNA libraries were prepared from selected samples using NEBNext Small RNA Library Preparation kit and sequenced on a HiSeq4000, and the data was analysed, focusing on miRNA and coding RNA reads. Results: Our data suggests distinct sources of variation in each biofluid. The cell type of origin was the strongest source of variability in cell culture supernatants, followed by RNA isolation method. Inn plasma/serum, RNA isolation method contributed the most to variability, suggesting enrichment of certain subsets of miRNAs and mRNAs by each method. In bile, the rather small number of miRNAs detected were reproducibly measured in samples isolated using the miRCury Biofluids kit, while fragments of many coding RNAs were efficiently isolated using all the tested methods. Summary/Conclusion: Our results demonstrate that reproducibility within and agreement between methods vary significantly across exRNA isolation methods and biofluids. Notably, none of the tested RNA isolation methods provided complete isolation of all exRNAs. Each method has a specific bias for specific exRNA carriers. These findings suggest that the selection of the method used for exRNA isolation is a critical consideration for studies in this field.

• Publisher: John Wiley & Sons, Inc
• Subject: Bile / Cell culture / Cell signaling / Data processing / Membrane filtration / Methods / miRNA / Purification / Ultracentrifugation