This decision tree allows a beginner to understand the best approaches available in the field to study extracellular RNA. The full list of protocols developed by the Extracellular RNA Communication consortium is available on the exRNA Portal at http://exRNA.org/resources/protocols, and at the Nature Protocol Exchange (http://www.nature.com/protocolexchange/labgroups/62073).

Note that many questions remain on how best to perform these procedures, so this decision tree is only a preliminary guideline to help you get started.

Collection and analysis of extracellular RNA consists of 5 major steps:
          1) Sample Collection
          2) Isolation and Enrichment of Extracellular Vesicles (optional)
          3) RNA Isolation
          4) Validation and Quantification
          5) RNA Sequencing

All of these protocols are strongly influenced by sample type and purity. Whichever methods you decide to use, it is vitally important to collect your samples (study and controls) using the exact same procedure. All heparin containing tubes should be avoided because heparin is a strong inhibitor of reverse transcriptase and PCR reactions, as well as EV uptake experiments.

The figure below, focused on processing serum, shows that some protocols in this collection cover more than one of the first three steps.

This version (1.3) of the exRNA protocols decision tree was last updated on 2016-08-10.