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DNA in fresh urine supernatant is not affected by additional centrifugation and is protected against deoxyribonuclease.

Author(s): Janovičová Ľ, Kmeťová K, Tóthová Ľ, Vlková B, Celec P

Publication: Mol Cell Probes, 2023, Vol. 68, Page 101900

PubMed ID: 36764623 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the amount of total, nuclear, and mitochondrial (mt) cell-free DNA (cfDNA) and the percentage of the total, nuclear, and mitochondrial cfDNA that was protected against DNase in whole urine, urine centrifuged once, urine centrifuged twice, and the resulting pellet. Midstream urine was collected from fifteen healthy volunteers. Urine was aliquoted immediately after collection. Half of the aliquots were treated with DNase (30 min at 37°C) while the other half were left untreated.  All aliquots were then left uncentrifuged, centrifuged once at 1600 g at 4°C for 10 min, or centrifuged at 1600 g at 4°C for 10 min followed by 16000 g at 4°C for 10 min. DNA was extracted with the QIAamp DNA Mini Kit and quantified with the High Sensitivity dsDNA Quantitation Kit. Nuclear DNA was quantified by real-time PCR amplification of β-globin and mitochondrial DNA by real-time PCR amplification of D-loop.

   Summary of Findings:

   Total cfDNA and mitochondrial cfDNA (mtcfDNA) were detected in all specimens from all 15 individuals, but nuclear cfDNA was only detected in all aliquots of 3 individuals.  The total DNA concentration in whole urine was highly variable with a coefficient of variance (CV) of 118%, but this was reduced to 103% when aliquots were centrifuged at 1600 g, and to 75% after aliquots were centrifuged twice.  The CV of cfDNA in the resulting pellet was 107%. Total cfDNA, nuclear cfDNA, and mtcfDNA were 66%, 88%, and 96% lower, respectively, in aliquots centrifuged once than those that were not centrifuged; but, only the decrease in mtcfDNA was significant (P<0.05). Total cfDNA, nuclear cfDNA, and mtcfDNA were all significantly lower in aliquots that were centrifuged twice than those that were not centrifuged (P<0.05, P<0.05 and P<0.001, respectively).  Seventy-four percent of the cfDNA in whole urine, 116% of cfDNA in urine centrifuged once, 86% of cfDNA in urine centrifuged twice, and 128% of cfDNA in the pellet was protected from DNase. In whole urine, a higher percentage of mtcfDNA than nuclear cfDNA was protected from DNase (173% versus 69%, respectively). The percentage of protected urinary mtcfDNA (from DNase) was also high in urine centrifuged once (287%) but not urine centrifuged twice (60%) or the resulting pellet (49%).

    

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Nucleic acid digestion	DNase treated Untreated
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared Not centrifuged

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