FIGURE 2.

Assays of miRNA transport. Currently, platelet-mediated transfer of miRNAs is studied in vitro and in vivo with various molecular and genetic tools that allow targeting of platelets, recipient cells and miRNAs: Platelets can be loaded with synthetic or labeled miRNAs, microvesicle (MV) release can be blocked with brefeldin A and platelet RNA can be degraded via RNase treatment. Moreover, the megakaryoblast cell line Meg-01 allows in vitro production of genetically modified platelets. In recipient cells, uptake of platelet-derived miRNAs can be blocked by specific inhibitors (e.g., targeting of PLA2IIA) and binding of miRNAs to the recipient cell RNA can be prevented by co-incubation with short target site-specific anti-sense antagomiRs or miRNA sponges, which harbor multiple miRNA target sites. Furthermore, blocking de novo miRNA synthesis in recipient cells allows to distinguish between endogenous and transferred miRNAs and the inhibitory potential of miRNAs can be quantified by reporter assays. In vivo studies take advantage of genetically or pharmacologically modified mice to study in vivo miRNA transfer.