Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

Figures & data

Table I 2012 ISEV Research Seminar discussion topics

Round table	Topic	Description
1	EV isolation	Pelleting, filtration, density gradient, controls
2	RNA isolation	Comparison of different isolation protocols
3	evRNA analysis	Hybridization, qPCR, RNase treatment
4	evRNA analysis and bioinformatics	Deep sequencing, analysis pipelines, recent developments
5	RNA loading and engineered vesicles	Electroporation, loading control, cellular production, targeting
6	Analysis of functional evRNA transfer	Reporter systems, physiological read-out systems

Table II Recommendations: resource development

Resource	Rationale	Details	Resource availability
Centralized normative control biofluid bank	For rigorous comparison of EV isolation methods, inter-study comparisons, and well-controlled biomarker development	Well-characterized normative matched biofluid samples (e.g. blood, CSF, urine, saliva) with “full disclosure” medical information and other details	Distribution by NIH or other repository; researchers would submit project-specific proposals for access
Artificial EV spike-in standards	To standardize recovery of EV and evRNA from biofluids; to facilitate independent verification of results	Four or five distinct vesicle sizes, closely mimicking bioparticles; loaded or not with markers (potentially inducible?): fluorescent protein or specific RNA sequences	Commercial or distributed by a repository to consortium members; established laboratories would be available for validation studies
Additional funding mechanisms for technique and normative control development	Disparity of results is dependent in part on procedural differences that could be resolved	Examples: head-to-head EV isolation comparisons; reproducibility of EV recovery throughout the day; sample volume and recovery studies; methods for determination of cellular/tissue origin of circulating EV	Standard grant application procedures
Collaborative sample and resource sharing network	Many groups focus on 1 particular type or size of EV or RNA; studies of different EV and evRNAs in the same samples would be useful	Participants could analyze different aliquots of the same biological fluids or send EV/fluids for analysis by laboratories with different focus/expertise	Website and voluntary cooperation