EV purification: enrichment of EVs/exosomes can be performed using size exclusion chromatography (SEC), ultracentrifugation, and precipitation. SEC-based purification can deliver both EVs and protein complexes to study presence of RNA in both compartments in parallel.
EV characterization: we use Nanoparticle Tracking Analysis (NTA), cryo-electron microscopy, as well as multiplex flow cytometry to determine EV size, concentration, nucleic acid content (SYBR Gold), and surface epitopes (CD81, CD63, CD9). Upon request, we can develop assays for analyzing other surface epitopes.
Non-specific characterization of EV cargo: we apply highly sensitive methods for protein, dsDNA, and ssRNA quantification in EV preparations to characterize their protein and nucleic acid content. RNase/DNase/Protease treatments are used to reduce noise from surface decorations with RNA/DNA.
exRNA analysis in EVs: we have developed robust protocols for small RNAseq, RNAseq, and RT-qPCR analysis of EVs (and protein complexes) to perform highly quantitative analysis of microRNA, mRNA, and other non-coding RNAs.
Our experience: we have completed more small RNA sequencing analyses of > 500 EV samples obtained from various biological samples. We have developed surface epitope staining protocols and have established fluorescence-based techniques for staining intracellular RNA/DNA content using NTA Quatt analysis as a means of EV manufacturing quality control.