Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data

Abstract

Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription‐quantitative PCR (RT‐qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV‐associated genes by RT‐qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human‐derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV‐associated RGs are stably detected in a size‐range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV‐associated RGs to enable normalization and thus steer the implementation of RT‐qPCR for the analysis of EV‐associated RNA cargo for research or clinical applications.

Therapeutic Potential of Bovine Milk-Derived Extracellular Vesicles

“…However, the major defect of SEC compared to other isolation methods is that it can co-isolate particles in the same size range, since isolation is based on size. Thus, coupling SEC with another chromatography method would be an option to improve the purity and enrich the EV subsets, as a recent report highlights that the double-SEC method even improves the purity [33]. Density gradient ultracentrifugation is more reliable than conventional ultracentrifugation, since particles are separated based on the solution density and have a lesser effect on the EV integrity.…”