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Collection and Extraction of Saliva DNA for Next Generation Sequencing
Published on: August 27, 2014
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Pre-Analytical Factors Affecting Extracellular DNA in Saliva
Ľubica Janovičová1, Dominika Holániová1, Barbora Vlková1
1Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, 811 08 Bratislava, Slovakia.
Diagnostics (Basel, Switzerland)
|February 10, 2024
Summary
Saliva extracellular DNA (ecDNA) is a potential disease biomarker. Additional centrifugation significantly reduces salivary ecDNA concentration, while freezing has minimal impact, suggesting ecDNA associates with cell debris.
Area of Science:
- Molecular Biology
- Biochemistry
- Biomarker Discovery
Background:
- Salivary DNA is easily collected for genetic analysis.
- Extracellular DNA (ecDNA) in saliva shows potential as a biomarker for oral and systemic diseases.
- Pre-analytical sample processing impacts salivary ecDNA quantity, but effects are not well understood.
Purpose of the Study:
- To investigate the effects of centrifugation and freezing on salivary ecDNA concentration.
- To determine the optimal pre-analytical processing for preserving salivary ecDNA.
Main Methods:
- Unstimulated saliva samples collected from 15 healthy volunteers.
- Samples were subjected to initial centrifugation (1600 x g) and either frozen or processed fresh.
- A second high-speed centrifugation (16,000 x g) was performed on fresh and thawed samples.
- DNA was isolated from supernatants and quantified using fluorometry and real-time PCR (targeting nuclear and mitochondrial DNA).
Main Results:
- Freezing had a negligible effect on salivary ecDNA concentration.
- An additional centrifugation step significantly reduced ecDNA by over 97% in both fresh and frozen samples.
- Quantitative PCR confirmed substantial decreases in both nuclear and mitochondrial ecDNA sequences post-centrifugation.
Conclusions:
- Salivary ecDNA concentration is primarily affected by additional centrifugation, not sample freezing.
- The findings suggest salivary ecDNA is largely associated with cell debris and apoptotic bodies.
- Future research should focus on disease-specific salivary ecDNA fractions.
