All the participants were asked to refrain from eating, drinking, smoking, and using oral hygiene products for at least 1 h prior to sample collection. Unstimulated saliva samples were collected using Danasaliva sample collection kit (Danagene, Ref. No 0603.43) according to the manufacturer's instructions. The saliva samples were taken to laboratory where they were stored at room temperature until processed (sample processing time less than 5 days). Then, saliva was centrifuged at 2600g for 15 min at room temperature. The salivary supernatant was removed from the pellet and centrifuged again at 13 000g for 1 min (two times) to avoid cell contamination. Salivary supernatants were stored at −80°C until further analysis.
DNA from salivary supernatants was extracted with the QIAamp DNA Circulating Nucleic Acid Kit (Qiagen, Ref. No. 55114), according to the manufacturer's instructions. DNA samples were stored at −20°C until use. Total DNA from each sample was quantified using the Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, Agilent's TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) was used to assess the fragment distribution of the extracted salivary DNA through Agilent Genomic DNA Screen Tape assay (Agilent Technologies, Ref. No. 5067–5365), in order to get a visual approach of DNA integrity.
DNA from salivary supernatants was extracted with the QIAamp DNA Circulating Nucleic Acid Kit (Qiagen, Ref. No. 55114), according to the manufacturer's instructions. DNA samples were stored at −20°C until use. Total DNA from each sample was quantified using the Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, Agilent's TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) was used to assess the fragment distribution of the extracted salivary DNA through Agilent Genomic DNA Screen Tape assay (Agilent Technologies, Ref. No. 5067–5365), in order to get a visual approach of DNA integrity.