Investigating the long RNA cargo of extracellular vesicles
Extracellular vesicles (EVs) are small membrane enclosed structures that are secreted by all cell types and are known to circulate throughout the body in biofluids such as plasma, urine, and saliva. The RNA cargo of EVs is of interest because of its potential to communicate information about health and disease between cells and tissues. EVs can also be used as a potential tool for monitoring health and disease because they can travel from distal tissue and accumulate in accessible biofluids. While most RNA profiling from EV cargos has examined small RNA (<45 nts), the actual species and function of long RNAs inside of EVs is still largely unknown. Current methods for isolating EVs and extracting RNA may inadvertently co-isolate protein-associated non-vesicular RNA and membrane bound RNA that are also present in biofluids. To specifically characterize RNAs inside of EVs, and remove RNA from the outside of EVs, we isolated vesicles from plasma samples using two different methods (differential ultracentrifugation and ExoRNeasy Kit from Qiagen). We then RNase treated half of the vesicles and left the other half untreated. RNA was then extracted from the vesicles using the miRNeasy Kit from Qiagen, quantified, and sequenced. We compare whole transcriptome sequencing results from vesicular-RNA with RNA extracted from vesicles that did not receive an RNase treatment. Methods, preliminary data, and trouble-shooting strategies will be discussed.