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Saliva represents an ideal matrix for diagnostic biomarker development as it is readily available and requires no invasive collection procedures. However, salivary RNA is labile and rapidly degrades. Previous attempts to isolate RNA from saliva have yielded poor quality and low concentrations. Here we compare collection and processing methods and propose an approach for future studies. The effects of RNA stabilisers, storage temperatures, length of storage and fasting windows were investigated on pooled saliva samples from healthy volunteers. Isolated RNA was assessed for concentration and quality. Bacterial growth was investigated through RT-PCR using bacterial and human primers. Optimal conditions were implemented and quality controlled in a clinical setting. The addition of RNA later  increased mean RNA yield from 4912 ng/μl to 15,473 ng and RNA Integrity Number (RIN) from 4.5 to 7.0. No significant changes to RNA yield were observed for storage at room temperature beyond 1 day or at -80 °C. Bacterial growth did not occur in samples stored at ambient temperature for up to a week. There was a trend towards higher RNA concentration when saliva was collected after overnight fasting but no effect on RIN. In the clinic, RNA yields of 6307 ng and RINs of 3.9 were achieved, improving on previous reports. The method we describe here is a robust, clinically feasible saliva collection method using preservative that gives high concentrations and improved RINs compared to saliva collected without preservative.

An optimised saliva collection method to produce high-yield, high-quality RNA for translational research