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 preamplification 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.

Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5–40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 °C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5–8 h varying exposure times; second: varying dose rates of 0.5–8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in ≥ 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 °C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 °C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 °C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.

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