Dataset Information

Characterization of RNA in saliva.

ABSTRACT:

Background

We have previously shown that human mRNAs are present in saliva and can be used as biomarkers of oral cancer. In this study, we analyzed the integrity, sources, and stability of salivary RNA.

Methods

We measured the integrity of salivary RNA with reverse transcription followed by PCR (RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites into the oral cavity, we used RT-PCR analysis of salivary RNA from the 3 major salivary glands, gingival crevice fluid, and desquamated oral epithelial cells. We measured stability of the salivary beta-actin mRNA by RT-qPCR of salivary RNA incubated at room temperature for different periods of time. We measured RNA association with other macromolecules by filtering saliva through pores of different sizes before performing RT-qPCR. To assess RNA-macromolecule interaction, we incubated saliva with Triton X-100 for different periods of time before performing RT-qPCR.

Results

In most cases, we detected partial- to full-length salivary mRNAs and smaller amounts of middle and 3' gene amplicons compared with the 5'. RNA was present in all oral fluids examined. Endogenous salivary beta-actin mRNA degraded more slowly than exogenous beta-actin mRNA, with half-lives of 12.2 and 0.4 min, respectively (P <0.001). Salivary RNA could not pass through 0.22 or 0.45 mum pores. Incubation of saliva with Triton X-100 accelerated degradation of salivary RNA.

Conclusions

Saliva harbors both full-length and partially degraded forms of mRNA. RNA enters the oral cavity from different sources, and association with macromolecules may protect salivary RNA from degradation.

SUBMITTER: Park NJ

PROVIDER: S-EPMC7108156 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Publications

Characterization of RNA in saliva.

Park Noh Jin NJ Li Yang Y Yu Tianwei T Brinkman Brigitta M N BM Wong David T DT

Clinical chemistry 20060406 6

<h4>Background</h4>We have previously shown that human mRNAs are present in saliva and can be used as biomarkers of oral cancer. In this study, we analyzed the integrity, sources, and stability of salivary RNA.<h4>Methods</h4>We measured the integrity of salivary RNA with reverse transcription followed by PCR (RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites into the oral cavity, we used RT-PCR analysis of salivary RNA from the 3 major salivary glands, gingival crevice fluid, a ...[more]

PMID: 16601067

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Project description:Dengue viruses (DENV) are expanding global pathogens that are transmitted through the bite of mosquitoes, mostly Aedes aegypti. As RNA viruses, DENV rely on RNA-binding proteins (RBPs) to complete their life cycle. Alternatively, RBPs can act as restriction factors that prevent DENV multiplication. While the importance of RBPs is well-supported in humans, there is a dearth of information about their influence on DENV transmission by mosquitoes. Such knowledge could be harnessed to design novel, effective interventions against DENV. Here, we successfully adapted RNA-affinity chromatography coupled with mass spectrometry-a technique initially developed in mammalian cells-to identify RBPs in Ae. aegypti cells. We identified fourteen RBPs interacting with DENV serotype 2 3'UTR, which is involved in the viral multiplication and produces subgenomic flaviviral RNA (sfRNA). We validated the RNA affinity results for two RBPs by confirming that AePur binds the 3'UTR, whereas AeStaufen interacts with both 3'UTR and sfRNA. Using in vivo functional evaluation, we determined that RBPs like AeRan, AeExoRNase, and AeRNase have pro-viral functions, whereas AeGTPase, AeAtu, and AePur have anti-viral functions in mosquitoes. Furthermore, we showed that human and mosquito Pur homologs have a shared affinity to DENV2 RNA, although the anti-viral effect is specific to the mosquito protein. Importantly, we revealed that AeStaufen mediates a reduction of gRNA and sfRNA copies in several mosquito tissues, including the salivary glands and that AeStaufen-mediated sfRNA reduction diminishes the concentration of transmission-enhancing sfRNA in saliva, thereby revealing AeStaufen's role in DENV transmission. By characterizing the first RBPs that associate with DENV2 3'UTR in mosquitoes, our study unravels new pro- and anti-viral targets for the design of novel therapeutic interventions as well as provides foundation for studying the role of RBPs in virus-vector interactions.

Dataset Information

Characterization of RNA in saliva.

Background

Methods

Results

Conclusions

Publications

Characterization of RNA in saliva.

Similar Datasets

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