Importance of RNA isolation methods for analysis of exosomal RNA: Evaluation of different methods

Exosomes are small RNA containing vesicles of endocytic origin, which can take part in cell-to-cell communication partly by the transfer of exosomal RNA between cells. Exosomes are released by many cells and can also be found in several biological fluids including blood plasma and breast milk. Exosomes differ compared to their donor cells not only in size but also in RNA, protein and lipid composition. The aim of the current study was to determine the optimal RNA extraction method for analysis of exosomal RNA, to support future studies determining the biological roles of the exosomal RNA. Different methods were used to extract exosomal and cellular RNA. All methods evaluated extracted high quality and purity RNA as determined by RNA integrity number (RIN) and OD values for cellular RNA using capillary electrophoresis and spectrophotometer. Interestingly, the exosomal RNA yield differed substantially between the different RNA isolation methods. There was also a difference in the exosomal RNA patterns in the electropherograms, indicating that the tested methods extract exosomal RNA with different size distribution. A pure column based approach resulted in the highest RNA yield and the broadest RNA size distribution, whereas phenol and combined phenol and column based approaches lost primarily large RNAs. Moreover, the use of phenol and combined techniques resulted in reduced yield of exosomal RNA, with a more narrow size distribution pattern resulting in an enrichment of small RNA including microRNA. In conclusion, the current study presents a unique comparison of seven different methods for extraction of exosomal RNA. As the different isolation methods give extensive variation in exosomal RNA yield and patterns, it is crucial to select an isolation approach depending on the research question at hand.