Background Since the discovery more than half a century ago, cell-free DNA (cfDNA) has become an attractive objective in multiple diagnostic, prognostic, and monitoring settings. However, despite the increasing number of cfDNA applications in liquid biopsies, we still lack a comprehensive understanding of the nature of cfDNA including optimal assessment. In the presented study, we continued testing and validation of common techniques for cfDNA extraction and quantification (qRT-PCR or droplet digital PCR) of nuclear- and mitochondrial cfDNA (ncfDNA and mtcfDNA) in blood, using a piglet model of perinatal asphyxia to determine potential temporal and quantitative changes at the levels of cfDNA. Methods and Results Newborn piglets (n = 19) were either exposed to hypoxia (n = 11) or were part of the sham-operated control group (n = 8). Blood samples were collected at baseline (= start) and at the end of hypoxia or at 40–45 min for the sham-operated control group. Applying the qRT-PCR method, ncfDNA concentrations in piglets exposed to hypoxia revealed an increasing trend from 7.1 ng/ml to 9.5 ng/ml for HK2 (hexokinase 2) and from 4.6 ng/ml to 7.9 ng/ml for β-globulin, respectively, whereas the control animals showed a more balanced profile. Furthermore, median levels of mtcfDNA were much higher in comparison to ncfDNA, but without significant differences between intervention versus the control group. Conclusions Both, qRT-PCR and the droplet digital PCR technique identified overall similar patterns for the concentration changes of cfDNA; but, the more sensitive digital PCR methodology might be required to identify minimal responses.

Birth asphyxia is the leading cause of death and disability in young children worldwide. Long non-coding RNAs (lncRNAs) may provide novel targets and intervention strategies due to their regulatory potential, as demonstrated in various diseases and conditions. We investigated cardinal lncRNAs involved in oxidative stress, hypoxia, apoptosis, and DNA damage using a piglet model of perinatal asphyxia. A total of 42 newborn piglets were randomized into 4 study arms: (1) hypoxia–normoxic reoxygenation, (2) hypoxia–3 min of hyperoxic reoxygenation, (3) hypoxia–30 min of hyperoxic reoxygenation, and (4) sham-operated controls. The expression of lncRNAs BDNF-AS, H19, MALAT1, ANRIL, TUG1, and PANDA, together with the related target genes VEGFA, BDNF, TP53, HIF1α, and TNFα, was assessed in the cortex, the hippocampus, the white matter, and the cerebellum using qPCR and Droplet Digital PCR. Exposure to hypoxia–reoxygenation significantly altered the transcription levels of BDNF-AS, H19, MALAT1, and ANRIL. BDNF-AS levels were significantly enhanced after both hypoxia and subsequent hyperoxic reoxygenation, 8% and 100% O2, respectively. Our observations suggest an emerging role for lncRNAs as part of the molecular response to hypoxia-induced damages during perinatal asphyxia. A better understanding of the regulatory properties of BDNF-AS and other lncRNAs may reveal novel targets and intervention strategies in the future.