Saliva, as a non-invasive and easily accessible biofluid, has been shown to contain RNA biomarkers for prediction and diagnosis of several diseases. However, systematic analysis done by our group identified two problematic issues not coherently described before: (1) most of the isolated RNA originates from the oral microbiome and (2) the amount of isolated human RNA is comparatively low. The degree of bacterial contamination showed ratios up to 1:900,000, so that only about one out of 900,000 RNA copies was of human origin, but the RNA quality (average RIN 6.7 + /- 0.8) allowed for qRT-PCR. Using 12 saliva samples from healthy donors, we modified the methodology to (1) select only human RNA during cDNA synthesis by aiming at the poly(A)+-tail and (2) introduced a pre-amplification of human RNA before qRT-PCR. Further, the manufacturer's criteria for successful pre-amplification (Ct values ≤ 35 for unamplified cDNA) had to be replaced by (3) proofing linear pre-amplification for each gene, thus, increasing the number of evaluable samples up to 70.6%. When considering theses three modifications unbiased gene expression analysis on human salivary RNA can be performed.

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

Department of Oncology and Radiotherapy University Hospital and Faculty of Medicine Hradec Kralove 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 Urology Bundeswehrkrankenhaus Ulm Ulm Germany

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

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

Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes