title: Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations.
abstract: Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm) and higher density (1.11 g/ml) than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively). Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.
authors.affiliation: Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.|Department of Oral and Maxillofacial Implantology, Tokyo Dental College, Tokyo, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Iwai
authors.affiliation: Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.
authors.forename: Tamiko
authors.initials: T
authors.lastname: Minamisawa
authors.affiliation: Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Suga
authors.affiliation: Department of Oral and Maxillofacial Implantology, Tokyo Dental College, Tokyo, Japan.
authors.forename: Yasutomo
authors.initials: Y
authors.lastname: Yajima
authors.affiliation: Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan; kshiba@jfcr.or.jp.
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Shiba
keywords: EVs; diagnosis; exosome; microRNA; saliva