Table 2.

Methods for determining EV-RNA purity and integrity.

Method	Use	Pros	Cons
Agilent Bioanalyzer chips	Integrity	Small volume required Highly sensitive Total length profile of RNA	Not suited for assessing small RNA integrity Assessment based on intact 18S/28S rRNAs generally depleted from EVs Sensitive to contaminants such as DNA
Next generation sequencing	Integrity & purity	Detects fragmentation, for example as 3′ bias in mRNA reads after poly-A selection Detects presence of foreign genetic material (e.g. derived from foetal bovine serum)	Erroneous assessment of fragments in the case of highly modified RNA types Long reads (i.e. PacBio) most useful but require lots of material
RT-PCR and derivatives (i.e. 5′/3′ RACE)	Integrity	Robust and sensitive, can map exact sites of fragmentation	Analysis of single transcripts only
Northern blot	Integrity	Robust and sensitive Simultaneous detection of full length and fragmented stretches of the same RNA	Analysis of single transcripts only Time-consuming
Proteinase-nuclease protection assay	Purity	Rigorously determine that RNA is present in EV lumen	Leftover nucleases may still be active at point of vesicle lysis
Blank run	Purity	Test kits and reagents for nucleic acid contamination	—
Picogreen	Purity	Test for presence of dsDNA	Not DNA-specific in samples with RNA concentrations over 130 ng ml–1