Overcoming challenges in human saliva gene expression measurements

0532064 - ÚJF 2021 RIV GB eng J - Journal Article
Ostheim, P. - Tichý, A. - Širák, I. - Davídková, Marie - Šťastná, M. - Kultová, G. - Paunesku, T. - Woloschak, G. - Majewski, M. - Port, M.
Overcoming challenges in human saliva gene expression measurements.
Scientific Reports. Roč. 10, č. 1 (2020), č. článku 11147. ISSN 2045-2322. E-ISSN 2045-2322
Institutional support: RVO:61389005
Keywords : RNA * blood stains * biomarkers
OECD category: Biophysics
Impact factor: 4.380, year: 2020
Method of publishing: Open access
https://doi.org/10.1038/s41598-020-67825-6

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.
Permanent Link: http://hdl.handle.net/11104/0310671