Abstract 2114: Salivary glands shedding RNA into saliva: Challenges and opportunities
Authors: Palmieri Laura , De Lima Dieila Giomo , Schubert Adrian D. , Ghantous Yasmin , Zamuner Fernando T. , Wysocki Piotr T. , Brait Mariana 
Published: June 2023

Abstract
Liquid biopsies hold great promises as non-invasive, reproducible, cost-effective and accurate cancer screening techniques. Saliva is attractive due its simple, virtually risk-free collection. The oral cavity and its surroundings may shed contents to saliva and it is essential to choose specific alterations that can indicate cells of origin. Common confounding contaminants may include bacteria and food debris. This study aims to test different saliva collection and processing protocols to build a path for saliva-based RNA cancer markers, by improving RNA specificity for tissue of origin (here salivary gland) and by increasing the quality of saliva extracted RNA. Saliva collection from 3 healthy subjects was performed with 5 different methodologies: gargle, non-stimulation, stimulation with gum, tabasco or ascorbic acid. These samples were protected with sterile 0.9% Sodium Chloride solution, QIAzol (Qiagen), RNAprotect Cell Reagent (Qiagen) or OrageneRNA (DNA Genotek), or without a protection step. RNA was then extracted with 4 different kits mirVana (ThermoFisher), QIAzol (Qiagen), RNEasy (Qiagen) and TRIzol Reagent (ThermoFisher). Total RNA was converted to cDNA using qScript cDNA (QuantaBio). RNA quality was evaluated on Agilent’s 2100 Bio analyzer. RNA quantity was evaluated with RiboGreen Reagent (Invitrogen) on SpectraMax M2 (Molecular Devices). RNA specificity for salivary gland origin was assessed by qRT-PCR for 2 genes HTN3 and CA6, which are specifically highly expressed in salivary glands, normalized by GAPDH. Quantity assessment showed an increased amount of RNA collected from unstimulated samples when comparing collection methods. For gargle, unstimulated and tabasco groups had higher RNA yields with the pellet fraction, mirVana extraction protocol and Qiazol protection. For all collection methods, we observed the best quality with the whole fraction, RNEasy extraction reagent, except for gargle. RNA protect showed the highest RNA quality, except for tabasco stimulated samples. There was no variation for gene stability considering HTN3 (mean 30.97/SD 1.37) and CA6 (mean 34.97/SD 0.54) CTs. HTN3 amplified earlier and more consistently than CA6 gene. Analyzing by collection method, the whole fraction was the most specific by gargle and unstimulated samples. mirVana was the best performing extraction method shared between all collection protocols, expect for ascorbic acid saliva stimulation. And Qiazol was the best protection methodology. We obtained the best RNA quality from whole saliva fraction using the RNA protect method and RNEasy extraction protocol. Our findings may build the foundation for desperately needed molecular markers for deadly diseases related to the salivary glands such as salivary gland tumors, aiming for early detection and patient surveillance. Our results also support further validation of strategies to assess saliva as a promising liquid biopsy fluid option.
Citation Format: Laura Palmieri, Dieila Giomo De Lima, Adrian D. Schubert, Yasmin Ghantous, Fernando T. Zamuner, Piotr T. Wysocki, Mariana Brait. Salivary glands shedding RNA into saliva: Challenges and opportunities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2114.