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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 RNAlater 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.

A variety of models have been proposed to explain regions of recurrent somatic copy number alteration (SCNA) in human cancer. Our study employs Whole Genome DNA Sequence (WGS) data from tumor samples (n = 103) to comprehensively assess the role of the Knudson two hit genetic model in SCNA generation in prostate cancer. 64 recurrent regions of loss and gain were detected, of which 28 were novel, including regions of loss with more than 15% frequency at Chr4p15.2-p15.1 (15.53%), Chr6q27 (16.50%) and Chr18q12.3 (17.48%). Comprehensive mutation screens of genes, lincRNA encoding sequences, control regions and conserved domains within SCNAs demonstrated that a two-hit genetic model was supported in only a minor proportion of recurrent SCNA losses examined (15/40). We found that recurrent breakpoints and regions of inversion often occur within Knudson model SCNAs, leading to the identification of ZNF292 as a target gene for the deletion at 6q14.3-q15 and NKX3.1 as a two-hit target at 8p21.3-p21.2. The importance of alterations of lincRNA sequences was illustrated by the identification of a novel mutational hotspot at the KCCAT42, FENDRR, CAT1886 and STCAT2 loci at the 16q23.1-q24.3 loss. Our data confirm that the burden of SCNAs is predictive of biochemical recurrence, define nine individual regions that are associated with relapse, and highlight the possible importance of ion channel and G-protein coupled-receptor (GPCR) pathways in cancer development. We concluded that a two-hit genetic model accounts for about one third of SCNA indicating that mechanisms, such haploinsufficiency and epigenetic inactivation, account for the remaining SCNA losses.