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Overcoming challenges in human saliva gene expression measurements
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SYSNO ASEP 0532064 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Overcoming challenges in human saliva gene expression measurements Author(s) Ostheim, P. (DE)
Tichý, A. (CZ)
Širák, I. (CZ)
Davídková, Marie (UJF-V) RID, ORCID, SAI
Šťastná, M. (CZ)
Kultová, G. (CZ)
Paunesku, T. (US)
Woloschak, G. (US)
Majewski, M. (DE)
Port, M. (DE)Number of authors 10 Article number 11147 Source Title Scientific Reports. - : Nature Publishing Group - ISSN 2045-2322
Roč. 10, č. 1 (2020)Number of pages 12 s. Publication form Print - P Language eng - English Country GB - United Kingdom Keywords RNA ; blood stains ; biomarkers Subject RIV BO - Biophysics OECD category Biophysics Method of publishing Open access Institutional support UJF-V - RVO:61389005 UT WOS 000562324300020 EID SCOPUS 85087654052 DOI 10.1038/s41598-020-67825-6 Annotation 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. Workplace Nuclear Physics Institute Contact Markéta Sommerová, sommerova@ujf.cas.cz, Tel.: 266 173 228 Year of Publishing 2021 Electronic address https://doi.org/10.1038/s41598-020-67825-6
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