Preservation of saliva for diagnostics

Saliva contains a wide range of proteins, antibodies and nucleic acids, which makes it an important diagnostic fluid in assessing oral and systemic diseases. The collection of saliva is convenient, non-invasive and cost effective. Objective: This study investigates different methods of collecting, preserving, and storing saliva to ensure simple collection and minimise degradation of proteins and nucleic acids. Methods: Unstimulated whole mouth saliva was collected from healthy volunteers into universal tubes either directly on ice or at room temperature. Saliva was divided, one batch was clarified by centrifugation and the other batch remained un-clarified. The saliva samples were then immediately stored under different conditions; (1) overnight storage at room temperature, 4°C or below 0°C; (2) overnight storage in the presence of protease inhibitor cocktail. Protein composition was analysed by SDS-PAGE and visualised with coomassie brilliant blue. Saliva was also treated with a range of different RNA preservatives, used according to the manufacturer's protocol. RNA was isolated using an RNeasy Micro Kit (Qiagen Inc) according to manufacturer's protocol and was quantified using Quant-iT™ Ribogreen® RNA kit (Molecular Probes, Invitrogen). Results: Collecting saliva on ice retained salivary proteins, in particular histatins and other low molecular weight proteins. Storing saliva samples at -20°C appeared to be optimal for preservation of salivary proteins with minimal loss. The use of protease inhibitor cocktail also preserved proteins. The yield of salivary RNA differed for each RNA preservative. RNA isolated from cell free was approximately 1ng/ml whilst un-clarified saliva was approximately 100 fold greater. Conclusions: Collecting saliva on ice and freezing at -20°C is a practical, quick and easy method for preserving saliva samples that can be implemented in a clinical setting. RNA in saliva can be quantified by fluorescence assay, although cell free RNA appears to be very low.