Number of the records: 1
Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes
- 1.
SYSNO ASEP 0554844 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes Author(s) Ostheim, P. (DE)
Alemu, S. W. (DE)
Tichý, A. (CZ)
Davídková, Marie (UJF-V) RID, ORCID, SAI
Šťastná, M. (CZ)
Kultová, G. (CZ)
Schuele, S. (DE)
Paunesku, T. (US)
Woloschak, G. (US)
Ghandhi, S. A. (US)
Amundson, S. A. (US)
Haimerl, M. (DE)
Stroszczynski, C. (DE)
Port, M. (DE)
Abend, M. (DE)Number of authors 15 Article number 2312 Source Title Scientific Reports. - : Nature Publishing Group - ISSN 2045-2322
Roč. 12, č. 1 (2022)Number of pages 13 s. Publication form Print - P Language eng - English Country DE - Germany Keywords RNA ; diagnostics ; biomarkers OECD category Biophysics Method of publishing Open access Institutional support UJF-V - RVO:61389005 UT WOS 000754021000035 EID SCOPUS 85124501125 DOI 10.1038/s41598-022-05670-5 Annotation Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P <= 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values <= 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva. Workplace Nuclear Physics Institute Contact Markéta Sommerová, sommerova@ujf.cas.cz, Tel.: 266 173 228 Year of Publishing 2023 Electronic address https://doi.org/10.1038/s41598-022-05670-5
Number of the records: 1