Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.

Biomedical Research Centre University Hospital Hradec Králové Czech Republic

Bundeswehr Institute of Radiobiology affiliated to the University of Ulm Neuherbergstr 11 80937 Munich Germany

Center for Radiological Research Columbia University Irving Medical Center New York NY 10032 USA

Department of Oncology and Radiotherapy University Hospital and Medical Faculty in Hradec Kralove Hradec Králové Czech Republic

Department of Radiation Dosimetry Nuclear Physics Institute of the Czech Academy of Sciences Prague Czech Republic

Department of Radiation Oncology Northwestern University Chicago IL 60611 USA

Department of Radiobiology Faculty of Military Health Sciences in Hradec Kralove University of Defence in Brno Brno Czech Republic

Department of Radiology University Hospital Regensburg Regensburg Germany

Institute for Hematology and Blood Transfusion Hospital Na Bulovce Prague Czech Republic

See more in PubMed

Quinn JF, et al. Extracellular RNAs: Development as biomarkers of human disease. J. Extracell. Vesicles. 2015;4:27495. doi: 10.3402/jev.v4.27495. PubMed DOI PMC

Li MH, Fu SB, Xiao HS. Genome-wide analysis of microRNA and mRNA expression signatures in cancer. Acta Pharmacol. Sin. 2015;36:1200–1211. doi: 10.1038/aps.2015.67. PubMed DOI PMC

Condrat CE, et al. miRNAs as biomarkers in disease: Latest findings regarding their role in diagnosis and prognosis. Cells. 2020;9:276. doi: 10.3390/cells9020276. PubMed DOI PMC

Szabo, D. T. Transcriptomic biomarkers in safety and risk assessment of chemicals. In Biomarkers in Toxicology (Ed. Gupta, R. C.) 1033–1038. 10.1016/B978-0-12-404630-6.00062-2 (Elsevier Inc, 2014).

Maron JL, et al. Neonatal salivary analysis reveals global developmental gene expression changes in the premature infant. Clin. Chem. 2010;56:409–416. doi: 10.1373/clinchem.2009.136234. PubMed DOI PMC

Watanabe K, Akutsu T, Takamura A, Sakurada K. Practical evaluation of an RNA-based saliva identification method. Sci. Justice. 2017;57:404–408. doi: 10.1016/j.scijus.2017.07.001. PubMed DOI

Lacombe J, et al. Analysis of saliva gene expression during head and neck cancer radiotherapy: A pilot study. Radiat. Res. 2017;188:75–81. doi: 10.1667/RR14707.1. PubMed DOI PMC

Ghizoni JS, Nichele R, de Oliveira MT, Pamato S, Pereira JR. The utilization of saliva as an early diagnostic tool for oral cancer: microRNA as a biomarker. Clin. Transl. Oncol. 2020;22:804–812. doi: 10.1007/s12094-019-02210-y. PubMed DOI

Li Y, et al. Salivary transcriptome diagnostics for oral cancer detection. Clin. Cancer Res. 2004;10:8442–8450. doi: 10.1158/1078-0432.CCR-04-1167. PubMed DOI

Kaczor-Urbanowicz KE, et al. Saliva diagnostics—Current views and directions. Exp. Biol. Med. 2017;242:459–472. doi: 10.1177/1535370216681550. PubMed DOI PMC

Chen W, Cao H, Lin J, Olsen N, Zheng SG. Biomarkers for primary Sjögren’s syndrome. Genomics Proteomics Bioinform. 2015;13:219–223. doi: 10.1016/j.gpb.2015.06.002. PubMed DOI PMC

Michael A, et al. Exosomes from human saliva as a source of microRNA biomarkers. Oral Dis. 2010;16:34–38. doi: 10.1111/j.1601-0825.2009.01604.x. PubMed DOI PMC

Yoshizawa JM, et al. Salivary biomarkers: Toward future clinical and diagnostic utilities. Clin. Microbiol. Rev. 2013;26:781–791. doi: 10.1128/CMR.00021-13. PubMed DOI PMC

Segal A, Wong DT. Salivary diagnostics: Enhancing disease detection and making medicine better. Eur. J. Dent. Educ. 2008;12:22–29. doi: 10.1111/j.1600-0579.2007.00477.x. PubMed DOI PMC

Ostheim P, et al. Overcoming challenges in human saliva gene expression measurements. Sci. Rep. 2020;10:11147. doi: 10.1038/s41598-020-67825-6. PubMed DOI PMC

Only, U. Oragene ® • RNA purification protocol using the Qiagen RNeasy Micro Kit for volumes up to 1000 µL. 3–5 (2012).

Life Technologies. mirVanaTM miRNA Isolation Kit. 33 (2011).

Applied Biosystems. High Capacity cDNA Reverse Transcription Kits for 200 and 1000 Reactions Protocol (Rev E). Manual 1–29 (2010).

RevTsc, T. SuperScript III Reverse Transcriptase (200U/µL) #18080-085. Manual 1–4 (2004).

Fisher, T. & July, S. TaqMan PreAmp Master Mix User Guide (2018).

Aro K, Wei F, Wong DT, Tu M. Saliva liquid biopsy for point-of-care applications. Front. Public Health. 2017;5:77. doi: 10.3389/fpubh.2017.00077. PubMed DOI PMC

Pernot E, Cardis E, Badie C. Usefulness of saliva samples for biomarker studies in radiation research. Cancer Epidemiol. Biomark. Prev. 2014;23:2673–2680. doi: 10.1158/1055-9965.EPI-14-0588. PubMed DOI

Dheda K, et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques. 2004;37:112–119. doi: 10.2144/04371RR03. PubMed DOI

Palanisamy V, Wong DT. Transcriptomic analyses of saliva. Methods Mol. Biol. 2010;666:43–51. doi: 10.1007/978-1-60761-820-1_4. PubMed DOI PMC

Navazesh M. Methods for collecting saliva. Ann. N. Y. Acad. Sci. 1993;694:72–77. doi: 10.1111/j.1749-6632.1993.tb18343.x. PubMed DOI

Patel RS, et al. High resolution of microRNA signatures in human whole saliva. Arch. Oral Biol. 2011;56:1506–1513. doi: 10.1016/j.archoralbio.2011.05.015. PubMed DOI PMC

Park NJ, et al. Salivary microRNA: Discovery, characterization, and clinical utility for oral cancer detection. Clin. Cancer Res. 2009;15:5473–5477. doi: 10.1158/1078-0432.CCR-09-0736. PubMed DOI PMC

Majem B, Li F, Sun J, Wong DTW. RNA sequencing analysis of salivary extracellular RNA. Methods Mol. Biol. 2017;1537:17–36. doi: 10.1007/978-1-4939-6685-1_2. PubMed DOI PMC

Tricarico C, et al. Quantitative real-time reverse transcription polymerase chain reaction: Normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal. Biochem. 2002;309:293–300. doi: 10.1016/S0003-2697(02)00311-1. PubMed DOI

Vandesompele J, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:1–12. doi: 10.1186/gb-2002-3-7-research0034. PubMed DOI PMC

Schmid H, et al. Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies. Kidney Int. 2003;64:356–360. doi: 10.1046/j.1523-1755.2003.00074.x. PubMed DOI

Applicability of Gene Expression in Saliva as an Alternative to Blood for Biodosimetry and Prediction of Radiation-induced Health Effects