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 ␤-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 fulllength 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 ␤-actin mRNA degraded more slowly than exogenous ␤-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 m 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.

Salivary mRNA targets for cancer diagnostics

“…We have observed that similar to plasma the endogenous cell-free mRNA in saliva is protected from immediate degradation [13,14]. Meanwhile, the salivary mRNA finding has been challenged by others unable to detect mRNA in saliva supernatant and also not in whole saliva [15].…”

“…Since our initial reports we have published and accumulated an abundance of additional supporting evidence, ranging from the characterization of salivary RNA [13], over assessing salivary RNA integrity by cDNA library analysis [21], to identifying and supporting the commercialization of an effective stabilizer of RNA in whole saliva, RNAprotect® Saliva (QIAGEN) [22]. Salivary cDNA library analysis showed that none of the 117 cloned mRNAs from saliva supernatant are from a pseudogene of contaminating genomic origin.…”

“…• Sources:The three major salivary glands, gingival crevice fluid, and dismantled oral epithelial cells contribute to the mRNA levels of whole saliva [13].…”

“…• Stability: We showed that RNAs in saliva, similar to cell-free RNA in plasma, are protected from immediate degradation by their association with macromolecules [13].…”

Endogenous small RNAs in grain: Semi-quantification and sequence homology to human and animal genes

“…The long history of consumption of rice as human food and the 10 results presented in the present manuscript together illustrate that small RNAs with perfect 11 complementarity to human genes are present in grain from a safely consumed staple crop, 12 therefore implying that dietary small RNAs do not exhibit oral activity to an extent that would 13 have a meaningful impact on human health. 14 15 Cooking and processing of foods may destroy endogenous RNA and DNA molecules, although 16 any remaining nucleic acids are likely to be digested after consumption due to low gastric pH 17 and due to pancreatic nucleases secreted into the intestine and those present in the saliva and 18 blood (Carver and Walker, 1995;Park et al, 2006). Less than 5% of nucleic acid catabolites are 19 used for de novo synthesis of nucleic acids and the remaining catabolites are utilized in other 20 biochemical reactions or undergo excretion in the urine and feces (Carver and Walker, 1995).…”