Quantification of circulating cell-free DNA (cfDNA) in urine using a newborn piglet model of asphyxia

Abstract

Cell free DNA (cfDNA) in plasma has been described as a potential diagnostic indicator for a variety of clinical conditions, including neonatal hypoxia. Neonatal hypoxia or perinatal asphyxia is a severe medical condition caused by a temporary interruption in oxygen availability during birth. Previously, we have reported temporal changes of cfDNA detected in blood in a newborn piglet model of perinatal asphyxia. However, cfDNA can also be found in other body liquids, opening for a less invasive diagnostic prospective. The objective of this study was to test and establish a reliable method for the isolation and quantification of cfDNA from urine and to explore changes in the quantities of cfDNA using a newborn piglet model of asphyxia. Animals were exposed to hypoxia-reoxygenation (n = 6), hypoxia-reoxygenation + hypothermia (n = 6) or were part of the sham-operated control group (n = 6) and urine samples (n = 18) were collected at 570 minutes post-intervention. Two alternative applications of cfDNA measurement were tested, an indirect method comprising a centrifugation step together with DNA extraction with magnetic beads versus a direct assessment based on two centrifugation steps. CfDNA concentrations were determined by a fluorescent assay using PicoGreen and by qRT-PCR. Genomic (gDNA) and mitochondrial DNA (mtDNA) cfDNA were determined in parallel, taking into account potential differences in the rates of damages caused by oxidative stress. In contrast to previous publications, our results indicate that the direct method is insufficient. Application of the indirect method obtained with the fluorescence assay revealed mean cfDNA levels (SD) of 1.23 (1.76) ng/ml for the hypoxia samples, 4.47 (6.15) ng/ml for the samples exposed to hypoxia + hypothermia and 2.75 (3.62) ng/ml for the control animals. The mean cfDNA levels in piglets exposed to hypoxia + hypothermia revealed significantly higher cfDNA amounts compared to mean cfDNA levels in the samples purely exposed to hypoxia (p < 0.05); however, no significant difference could be determined when compared to the control group (p = 0.09). Application of the indirect method by qRT-PCR revealed mean cfDNA levels of mtDNA and gDNA at the detection limit of the technique and thus no reliable statistics could be performed between the observed cfDNA levels in the investigated groups. The methodology for detection and monitoring of cfDNA in urine has to be further optimized before it can be applied in a clinical setting in the future.

Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxia

“…The assumption of a disease-dependent spread of DNA fragments is counteracted by the observation of base-line levels of cfDNA detected in the blood circulation of healthy individuals, with age, sex, hormonal, biorhythmic, and other dependencies [11]. Further, cfDNA fragments are not only circulating in blood, but can also be found in other body fluids, like saliva, urine, or cerebrospinal fluid [12][13][14].…”

“…Many different markers have been proposed to estimate the oxidative stress reactions and cell damages in its pathophysiological background; however, the key regulatory elements are still not identified [12]. Studies have proposed that cfDNA might be an early indicator of perinatal asphyxia [12,13]. Moreover, Tuaeva et al, 2008 observed that premature birth is associated with increased cfDNA concentrations in the blood of neonates [26].…”

“…Moreover, Tuaeva et al, 2008 observed that premature birth is associated with increased cfDNA concentrations in the blood of neonates [26]. These findings built the basis to investigate circulating cfNAs as a potential biomarker in the diagnostics of the damages caused by perinatal asphyxia [12,13]. But, the analysis of cfDNA is challenging, including the best choice of extraction processes, quantification techniques, and applications for the assessment of cfNAs [11,[27][28][29][30].…”

“…We have previously proposed a cellular mechanism of cfDNA production and discharge via the generation of reactive oxygen species (ROS) in both, the nucleus and in the mitochondria [ 12 , 13 ]. Nuclear and mitochondrial genomes are principally different, in terms of biological tasks, size, copy number, and organization, with striking consequences for cfDNA determination (Fig.…”

“…Assessment of changes in the amount of cfDNA in various body fluids has become an interesting tool for many diseases and conditions, including cancer, prematurity, or hypoxia. We have previously postulated [ 12 , 13 ], that the observed variations in cfDNA might be provoked by reactive oxygen species (ROS) damaging various cell structures. Consequently, fragments of both, nuclear and mitochondrial cfDNA (ncfDNA and mtcfDNA, respectively) may be released into the blood stream.…”