Saliva contains biological information as blood and is recognized as a valuable diagnostic medium for their noninvasiveness. Although “-omics” researches have tried to investigate saliva, the origin and significance of its contents are not clear, and its usage is largely confined to oral disease in the diagnostic and prognostic field.
In an attempt to broaden the applicability of saliva and to find systemic disease-derived RNA in saliva, researchers from the Pohang University of Science and Technology made mouse models that had human melanoma and isolated extracellular vesicles (EVs) from their saliva by an aqueous 2-phase system (ATPS), then identified and evaluated their expression of human melan-A RNA, which is associated with melanoma on skin. With ATPS, EVs were isolated efficiently and stably while taking less time compared to isolation by ultracentrifugation. When ATPS was used to isolate EVs from saliva, the mean ± SD percentage of EVs recovered from initial EVs was 38.22% ± 18.55% by the number of particles, and the mean ± SD percentage of RNA recovered from the initial amount was 60.33% ± 5.34%. RNAs within isolated EVs were analyzed subsequently by reverse transcription quantitative polymerase chain reaction and polymerase chain reaction from saliva and plasma. In melanoma mice, amplification of human melan-A was identified from saliva and plasma, even though a relative amount of normalized melan-A was lower than that of plasma. These results present a possibility that RNAs derived from systemic disease are transferred into saliva from blood in EVs. Also, they suggest that saliva could be exploited in obtaining information about systemic disease, not only about oral disease, by examining RNAs in EVs from saliva instead of blood.
Isolation of>extracellularvesicles (EVs) by the aqueous
2-phase system (ATPS) from precleaned saliva
(A) Schematic diagram of isolation process by ATPS. After phase separation, the upper polyethylene glycol (PEG) phase is rich in protein, and the lower dextran (DEX) phase is rich in EVs. (B) Comparison of recovered RNA amount and EV particle numbers by isolation methods. (C) Comparison of EV markers by isolation methods. EV samples, which were isolated from the same initial volume by ATPS or double pelleting, were loaded into each lane under the same condition. (D) Comparison of EV particle numbers per total protein amount by isolation methods. Protein amount was measured by the Bradford assay. (E) Comparison of size distribution of EVs by isolation method. Solid lines: average value at each size; gray area: gap between dotted lines that correspond to ± SD.