Poster Presentation Australasian Extracellular Vesicles Conference 2020

High precision isolation of extracellular vesicles and exRNA for clinical studies: A combination strategy of size exclusion chromatography and RNA extraction technologies  (#54)

Emma LCJ Blundell 1 , Anoop K Pal 2
  1. Izon Science Ltd, Christchurch, Canterbury , New Zealand
  2. Izon Science Ltd, Medford, Massachusetts, USA

Extracellular vesicles (EVs) derived from biological fluids possess extensive heterogeneity with regards to their size, number, membrane composition and cargo. Tremendous research interests lie toward development and use of EV fractions of bio-fluids as rich sources of robust diagnostic and prognostic biomarkers, more recently involving extracellular RNA (exRNA) such as micro RNA (miRNA). The ability to isolate EVs and exRNA subpopulations reproducibly from biological samples is a current challenge in EV research.

Size exclusion chromatography (SEC) is the most standardised technique for isolating EVs, already widely used in the field for biological samples. Significant improvements to SEC have been achieved through automating the process using the Automatic Fraction Collector (AFC) with advanced functionality that includes a precise mass-based measurement for the fractions, improves repeatability and speeds up workflow. SEC columns provide a convenient, reproducible and highly effective means of eliminating >99% of non-vesicular protein from bio-samples, effectively separating exosomal and non-exosomal volumes for further analysis, both important in biomarker research whilst maintaining EV structural integrity and biological function. Isolated samples can then be accurately characterised using tunable resistive pulse sensing (TRPS) where simultaneous size, concentration and zeta potential measurements can be completed. 

The qEV-AFC platform alone allows for QA, high-precision EV volume collection and minimises current reproducibility issues for clinical studies as well as retaining the functionality of the isolated molecules. Thus, a qEV-AFC-exRNA combination strategy to isolate EVs and exRNA from various biological fluids will result in reproducible high-purity samples and will be revolutionary for the EV-research demographic. The qEV-AFC-exRNA initiative allows for interlaboratory consistency and for users to complete more comprehensive analysis of a single sample, a key driver being the importance of exosomes and their cargo as diagnostic and prognostic markers in early stages of disease.

The ability to setup parallel AFC units allows for processing multiple samples simultaneously; a critical consideration for groups aiming at processing cohorts of samples as part of larger clinical trials. qEV-AFC-exRNA workflow is currently being tested with key academic groups; one such pilot study involves evaluating plasma derived exosomal miRNA obtained from cardiovascular patients (n=40).