Abstract:
: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.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Ostheim P,Tichý A,Sirak I,Davidkova M,Stastna MM,Kultova G,Paunesku T,Woloschak G,Majewski M,Port M,Abend Mdoi
10.1038/s41598-020-67825-6subject
Has Abstractpub_date
2020-07-07 00:00:00pages
11147issue
1issn
2045-2322pii
10.1038/s41598-020-67825-6journal_volume
10pub_type
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