The consortium has developed Standard Operating Procedures (SOPs) for purification of extracellular vesicles and RNA, as well as for exRNA sequencing and data analysis. A major goal of ERCC Stage 1 was to standardize these procedures to minimize experimental variation across labs and experiments.
Most of the protocols were updated after first release in 2015 based on vetting the originals in multiple labs. The major change to isolation and enrichment protocols was an increase in input volume of biofluid from 200 µL to 500 µL. The larger input volume yields more reproducible amounts of RNA isolation across multiple experiments.
Updated protocols are indicated here as version 2 (v2). We have maintained links to version 1 protocols for archival purposes.
miRDaR is a web application designed to help researchers select the best RNA isolation protocol for detection and reproducibility of miRNAs of interest to them. It accompanies the publication Srinivasan et al. Small RNA sequencing across diverse biofluids identifies optimal methods for exRNA isolation. Cell (2019) and replaces the earlier protocols decision tree, still linked here for archival purposes.
This decision tree allows a beginner to understand the best approaches available in the field to study extracellular vesicles and extracellular RNA. See this blog post for an in-depth discussion of how to select protocols from this page. The Protocols Decision Tree currently still refers to version 1 protocols.
Collection of Biofluids
Isolation and Enrichment of Extracellular Vesicles
All of the protocols below can be found in the Supplementary Appendix of the publication above.
Validation and Quantification
This publication includes four variants on the main idea of adding random adapters at the amplification stage of sequencing.
Proteomics on Extracellular Vesicles