Figure 2.
Stability of extracellular vesicle-associated miRNAs at physiological temperature. Extracellular vesicles (EVs) were isolated from C2C12 myotube conditioned medium (CM) by tangential flow filtration and size exclusion liquid chromatography. (a) 280 nm absorbance trace for liquid chromatography fractions. The pooled fractions which contain the EVs are indicated on the plot. Isolated EVs were analyzed by (b) nanoparticle tracking analysis, and (c) Western blot for exosome markers (ALIX, TSG101, and CD81). (d) Isolated EV or unfractionated CM samples were incubated at 37ºC and miRNA abundance determined at each time point by RT-qPCR. (e) Estimation of the proportion of ex-miRNAs that are unstable and non-vesicular (i.e. the amount of miRNA that is degraded after incubation at 37°C for 76 hours, from Figure 1), EV-associated (i.e. the proportion of ex-miRNA contained in the EV fractions after liquid chromatography), and the stable non-vesicular (i.e. the proportion of ex-miRNA remaining after incubation at 37°C for 76 hours minus the EV-associated fraction). Values are mean±SEM, n = 3.