Pre-Analytical Factors Affecting Extracellular DNA in Saliva.
Author(s): Janovičová Ľ, Holániová D, Vlková B, Celec P
Publication: Diagnostics (Basel), 2024, Vol. 14, Page
PubMed ID: 38337765 PubMed Review Paper? No
Purpose of Paper
This paper compared the concentration of total, nuclear, and mitochondrial extracellular DNA (cell-free DNA, cfDNA) and cfDNA fragment length in saliva specimens that were centrifuged once versus twice and/or frozen after the initial centrifugation or analyzed fresh. Potential effects of DNase treatment on cfDNA concentration and fragment sizes were also examined.
Conclusion of Paper
Statistical analysis (ANOVA) showed a significant effect of centrifugating saliva specimens twice versus once, but freezing after the first centrifugation did not affect cfDNA concentration. In fresh and frozen saliva specimens, respectively, only 2.25% and 1.65% of the cfDNA in the specimen centrifuged once at 1,600 g remained after a second centrifugation step at 16,000 g was performed. Importantly, the decrease in cfDNA that occurred after a second centrifugation included declines in both nuclear and mitochondrial DNA, but the ratio of mitochondrial DNA to nuclear DNA was unaffected by the second centrifugation. The concentrations of total nuclear or mitochondrial cfDNA were not correlated between specimens centrifuged once versus twice. While treatment with DNase I removed approximately 80% of total cfDNA in specimens that were centrifuged once, only nonsignificant decreases were observed after DNase treatment in saliva specimens that were centrifuged twice or in microparticles. The fragmentation profile of the cfDNA obtained from saliva specimens that were centrifuged once was long with no clear peaks, but one specimen pool displayed clear peaks at 400, 500, and 1000 bp after a second centrifugation, peaks that were also observed in the microparticles. Interestingly, all of the long DNA was removed after DNase treatment, but the peaks remained, which the authors attribute to the protection of a subset of cfDNA. Freezing saliva reduced DNase activity by 36%, but the concentrations of total, nuclear, and mitochondrial cfDNA were correlated between fresh and frozen saliva.
Studies
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Study Purpose
This paper compared the concentration of total, nuclear, and mitochondrial cfDNA and cfDNA fragment length in saliva specimens that were centrifuged once versus twice and/or frozen after the initial centrifugation or analyzed fresh. Potential effects of DNase treatment on cfDNA concentration and fragment sizes were also examined. Unstimulated saliva was collected from 15 healthy volunteers and was immediately aliquoted and centrifuged at 1600 g for 10 min at 4°C to remove cells. Aliquots were either used directly for cfDNA extraction; centrifuged again at 16,000 g at 4°C, frozen (temperature not specified) and used directly for cfDNA extraction; or frozen at (temperature not specified) and then centrifuged again at 16,000 g at 4°C. An aliquot of each saliva was treated with DNase1. cfDNA was isolated using the QIAamp DNA Mini Kit on a QIAcube instrument and quantified using the Qubit dsDNA High Sensitivity Assay Kit and by real-time PCR amplification of beta globin (nuclear) and D-loop (mitochondrial). The fragmentation profile was analyzed in two concentrated cfDNA pools (details not provided) using the Agilent High Sensitivity DNA Kit on a Bioanalyzer instrument. DNase activity was assessed using a single-radial enzyme-diffusion assay.
Summary of Findings:
The concentration of cfDNA was significantly affected (P<0.001) by the number of centrifugation steps (one versus two) but not by whether or not specimens were frozen after the initial centrifugation. In fresh and frozen specimens, respectively, only 2.25% (P<0.05) and 1.65% (P<0.01) of the cfDNA in the saliva specimen that was centrifuged once at 1,600 g remained after a second centrifugation step at 16,000 g was performed . Importantly, the decrease in cfDNA that occurred with a second centrifugation step included declines in both nuclear and mitochondrial DNA in fresh (P<0.01, both) and frozen specimens (P<0.05 and P<0.01, respectively), but the ratio of mitochondrial DNA to nuclear DNA was unaffected by a second centrifugation. DNase activity in saliva decreased by 36% in specimens that were frozen (P=0.005). The total, nuclear, and mitochondrial concentrations of cfDNA were correlated when fresh and frozen saliva specimens were compared (r=0.58, P=0.02; r=0.90, P<0.001; and r=0.78, P<0.001, respectively), but no correlation in the concentrations of total nuclear or mitochondrial cfDNA was observed between specimens centrifuged once versus twice. Treatment with DNase I, removed approximately 80% of total cfDNA in saliva specimens that were centrifuged once (P<0.01), but only led to nonsignificant decreases in saliva specimens that were centrifuged twice or in microparticles. When nuclear and mitochondrial cfDNA were examined, the effect of DNase I treatment was not significant, which the authors state may be due to the small sample size. The fragmentation profile of cfDNA obtained from saliva that was centrifuged once was long with no clear peaks, but one cfDNA pool displayed clear peaks at 400, 500, and 1000 bp after a second centrifugation, peaks that were also observed in the microparticles. Interestingly, all of the long DNA was removed after DNase treatment, but the peaks remained, which the authors attribute to protection of a subset of cfDNA.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)
Analyte Extraction and Purification Nucleic acid digestion Untreated
DNase I treated