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Methodological influences on circulating cell-free-mitochondrial and nuclear DNA concentrations in response to chronic stress.

Author(s): Daubermann C, Herhaus B, Neuberger EWI, Simon P, Petrowski K

Publication: Mol Biol Rep, 2025, Vol. 52, Page 303

PubMed ID: 40080226 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared cell-free mitochondrial DNA (cf-mtDNA) and nuclear DNA (cf-nDNA) levels that were quantified in (i) plasma directly versus after DNA isolation, and (ii) directly in plasma obtained by a single centrifugation at 600 x g and plasma obtained by centrifugation at 600 x g then 2,500 x g or 2,500 x g then 16,000 x g. The authors also investigated potential correlations between psychological stress scores and levels of cf-mtDNA and cf-nDNA. Blood was collected from 22 healthy volunteers (19 women and 3 men) into K₃EDTA tubes between 2 and 5 PM after 30 min of being stationary (reading) and 2 h without food or beverages. Plasma was obtained by serial centrifugation at 600 x g for 15 min, then 2,500 x g for 15 min, followed by 16,000 x g for 15 min, with a plasma aliquot removed after each centrifugation. Plasma was stored at -20°C. DNA was isolated using the QIAamp DNA Blood Mini Kit and stored at -20°C. cf-nDNA and cf-mtDNA were quantified directly in plasma and in extracted DNA by real-time PCR amplification of a 90 bp fragment of LINE1 and a 102 bp fragment of ND1, respectively. Prior to blood collection, psychological stress was evaluated using the Trier Inventory for Chronic Stress (TICS-9).

   Summary of Findings:

   DNA extraction (with the QIAamp DNA Blood Mini Kit) prior to amplification led to DNA loss with only 56.7% of the cf-mtDNA in plasma observed following extraction; extracted samples contained just 56.7% of the cf-mtDNA that was directly quantified in plasma. The amount of cf-mtDNA recovered was modestly correlated between matched plasma and DNA extracts (ρ=0.59, P=0.003).  Levels of cf-mtDNA in plasma (without DNA extraction) declined with each additional centrifugation step at a higher speed, with significantly higher levels of cf-mtDNA observed in plasma centrifuged once at 600 x g than in plasma centrifuged at 600 x g followed by 2,500 x g (P<0.001) and higher levels in plasma centrifuged at 600 x g followed by 2,500 x g than in plasma centrifuged at 600 x g followed by 2,500 x g and then 16,000 x g (P<0.001). Importantly, when the third centrifugation step at 16,000 x g was used to obtain plasma, cf-mtDNA levels dropped below the limit of quantification in 55% of specimens.  cf-nDNA levels decreased when plasma centrifugation at 600 x g was followed by centrifugation at 2,500 x g (P<0.001), but did not decrease further when a third centrifugation at 16,000 x g was performed. Psychological stress levels were negatively and modestly correlated with cf-mtDNA levels in plasma centrifuged at 16,000 x g (ρ=-0.51, P=0.015) but were not correlated with cf-nDNA levels in the same plasma specimens.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared Multiple speeds compared
   Real-time qPCR Specific	Targeted nucleic acid	LINE-1 (cf-nDNA) ND1 (cf-mtDNA)
   Preaquisition	Diagnosis/ patient condition	TICS score of 1-26
   Analyte Extraction and Purification	Analyte isolation method	No extraction performed Extracted with QIAamp DNA Blood Mini Kit

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