Abstract
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.
Highlights
Isolation of RNA from whole saliva, a non-invasive and 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
We identified two problematic issues not coherently described before when working with human whole saliva: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming[15]
The aim of this manuscript is to examine if there are any other prerequisites that have to be considered for satisfying results in this approach e.g. the optimal saliva sampling time or the impact of potential confounders such as demographics or epidemiological factors comprising social habits, oral hygiene or previous radiological diagnostic procedures
Summary
Isolation of RNA from whole saliva, a non-invasive and 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 the current work, we have modified the methodology to (1) select only human RNA during cDNA synthesis by targeting the poly(A)+-tailed mRNA and (2) introduced pre-amplification of human RNA before qRT-PCR15 The combination of these two important modifications to our protocols resulted in sufficient amounts of high quality and quantity human RNA. Not being a sterile medium like blood, saliva can be affected by many confounding variables, which influence the quality and quantity of RNA that can be isolated from human whole saliva The aim of this manuscript is to examine if there are any other prerequisites that have to be considered for satisfying results in this approach e.g. the optimal saliva sampling time or the impact of potential confounders such as demographics (e.g. sex, age) or epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash) or previous radiological diagnostic procedures (e.g. number of computed tomography/CTscans). We examined baseline gene expression values of ten commonly used housekeeping genes in human whole saliva to accomplish the above mentioned tasks and present our analysis here
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