Exosomes from human saliva as a source of microRNA biomarkers

Abstract

Objective: The aim of this study was to examine the presence of microRNAs (miRNAs) within exosomes isolated from human saliva and to optimize and test methods for successful downstream applications.

Design: Exosomes isolated from fresh and frozen glandular and whole human saliva were used as a source of miRNAs. The presence of miRNAs was validated with TaqMan quantitative PCR and miRNA microarrays.

Results: We successfully isolated exosomes from human saliva from healthy controls and a patient with Sjögren's syndrome. microRNAs extracted from the exosomal fraction were sufficient for quantitative PCR and microarray profiling.

Conclusions: The isolation of miRNAs from easily and non-invasively obtained salivary exosomes with subsequent characterization of the miRNA expression patterns is promising for the development of future biomarkers of the diagnosis and prognosis of various salivary gland pathologies.

Figure 1

(a) Western blot analysis of TSG101, a classic exosomal marker, of the exosomal lysates isolated from parotid saliva (40mg loaded sample) (B), from parotid saliva (20mg loaded sample) (C), submandibular saliva (D), frozen parotid saliva stored at −20°C for 7 days (E) and from frozen submandibular saliva stored at −20°C for 7 days (F). Negative control w as run in (A). (b) Average total RNA concentration per 100ul of saliva collected. Technical difficulties with the mucin content of saliva precludes a higher RNA concentration to be obtained. (c) A bioanalyzer profile of parotid saliva derived exosomal microRNAs. The enrichment in RNAs of the sizes of microRNAs is evident; 68% of the RNAs of size between 0 and 233 nucleotides falls within the microRNA range of 10–40 nucleotides and has an average size of 25 nucleotides.