Extraction and Analysis of Extracellular Vesicle-Associated miRNAs Following Antibody-Based Extracellular Vesicle Capture from Plasma Samples.

Extracellular vesicle (EV)-associated RNAs (EV-RNA) are under intense investigation due to their potential role in health and disease. Several approaches are currently employed to isolate blood-derived EVs for RNA analysis, most of which are either time-consuming and expensive, such as methods based on EVs physical properties (ultracentrifugation and Optiprep density gradient), or also copurify blood contaminants, mostly protein aggregates and immune complexes, (such as chemical precipitation). In addition, there is a lack of standardized protocols for the extraction of EV-RNA and very little consensus on the technological platforms and normalization tools for assessing the expression levels of different RNA species. These methodological issues complicate the comparison between independent data sets, potentially biasing results and conclusions.In this book chapter we propose a protocol that might overcome some of the abovementioned issues through antibody-based isolation of blood-derived EVs followed by extraction and expression analysis of small-RNA species (miRNA) by reverse transcriptase quantitative PCR (RT-qPCR). The advantages of immunoaffinity approaches over other isolation methods are multiple and include: (1) the selective enrichment of specific EV subpopulations with restricted tissue/cell origin, (2) reduction of matrix effects and blood contaminants that may confound miRNA profiling from complex biological fluids and (3) easy coupling to conventional quantitative assays (e.g., RT-qPCR). In conclusion, we describe a protocol for standard enrichment and quantitative analysis of EV-miRNAs from blood and we warrant for technological improvements, such as the use of novel biomaterials, surface chemistries, binding agents and assay/sensor design that may further improve it.