Pre-Analytical Factors Affecting Extracellular DNA in Saliva.

Salivary DNA is widely used for genetic analyses because of its easy collection. However, its extracellular fraction in particular, similar to the extracellular DNA (ecDNA) in plasma, could be a promising biomarker for oral or systemic diseases. In contrast to genetics, the quantity of salivary ecDNA is of importance and can be affected by the pre-analytical processing of samples, but the details are not known. The aim of our study was to analyze the effects of centrifugation and freezing of saliva on the concentration of ecDNA in saliva. Fifteen healthy volunteers, free of any known systemic or oral diseases, were asked to collect unstimulated saliva samples. Aliquots were centrifuged at 1600× and frozen or directly processed. The fresh or thawed cell-free saliva samples underwent subsequent centrifugation at 16,000× . The supernatants were used for DNA isolation and quantification using fluorometry and real-time PCR. While freezing had minimal effects on the salivary ecDNA concentration, another centrifugation step decreased ecDNA considerably in both fresh and frozen samples (by 97.8% and 98.4%, respectively). This was mirrored in the quantitative PCR targeting a nuclear (decrease by 93.5%) and mitochondrial (decrease by 97.7%) ecDNA sequence. In conclusion, in this first study focusing on the technical aspects of salivary ecDNA quantitation, we show that, regardless of its subcellular origin, the concentration of ecDNA in saliva is mainly affected by additional centrifugation and not by the freezing of centrifuged cell-free saliva samples. This suggests that most salivary ecDNA likely is associated with cell debris and apoptotic bodies. Which fraction is affected by a particular disease should be the focus of further targeted studies.