Overcoming challenges in human saliva gene expression measurements

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 originat...

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Published in:Scientific reports 2020-07, Vol.10 (1), p.11147-11147, Article 11147
Main Authors: Ostheim, Patrick, Tichý, Ales, Sirak, Igor, Davidkova, Marie, Stastna, Marketa Markova, Kultova, Gabriela, Paunesku, Tatjana, Woloschak, Gayle, Majewski, Matthaeus, Port, Matthias, Abend, Michael
Format: Article
Language:English
Subjects:
631/337
631/337/2019
Adult
Contamination
DNA, Complementary - genetics
Female
Gene Expression
Gene Expression Profiling - methods
Humanities and Social Sciences
Humans
Male
Microbiomes
multidisciplinary
Polyadenine
Real-Time Polymerase Chain Reaction - methods
Ribonucleic acid
RNA
RNA - analysis
RNA, Bacterial - genetics
RNA, Ribosomal, 18S - genetics
Saliva
Saliva - chemistry
Saliva - metabolism
Science
Science (multidisciplinary)
Transcription
Transcriptome
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-020-67825-6