Analysis of cf-mtDNA and cf-nDNA fragment size distribution using different isolation methods in BV-2 cell supernatant of starvation-induced autophagy.

Fragment size distribution, the important biological properties of cell-free DNA (cfDNA), provides useful information required for diagnostic assay development. However, besides methodological discrepancies, it varies due to the complicated origins and occurrences of in vivo cfDNA. In addition, limited data are available concerning the cfDNA associated with autophagy and distributional difference between cf-mitochondrial DNA (cf-mtDNA) and cf-nuclear DNA (cf-nDNA) fragments. Here we developed an in vitro model of mouse microglial cell (BV-2) with starvation-induced autophagy, in which cfDNA was isolated from the cell supernatant by ultrafiltration (UF) and column-based commercial kit (CC), respectively. Using Agilent 2100 Bioanalyzer, a DNA ladder pattern as the presence of peaks corresponding to mono-, di- and tri-nucleosomes was clearly visualized both in isolation products of UF and CC. However, we also detected shorter fragments than mono-nucleosome by UF. In comparing the UF and CC, we found that the former produced the higher recovery efficiency for spiked-in DNA of shorter fragments than mono-nucleosome in both water and medium, but the latter was superior for spiked-in DNA fragments which were longer than or equal to mono-nucleosome in medium. Combined with these two isolation methods, we have observed that autophagy-associated cf-mtDNA and cf-nDNA were both highly enriched in <mono-nucleosomes fragments more than 71%, and showed no significant differences in the relative percentages for these four fragment sizes. These results have improved our understanding of the fragment size distribution of autophagy-derived cf-mtDNA and cf-nDNA in vitro, and might further develop application of cfDNA as a diagnostic tool.

Gong Y ，Huang Q ，Deng Y ，Zhou L ，Yi X ，Zhu J ，Wu W ... -  《-》

Comparison of paired cerebrospinal fluid and serum cell-free mitochondrial and nuclear DNA with copy number and fragment length.

Most studies on cell-free DNA (cfDNA) were only for single body fluids; however, the differences in cfDNA distribution between two body fluids are rarely reported. Hence, in this work, we compared the differences in cfDNA distribution between cerebrospinal fluid (CSF) and serum of patients with brain-related diseases. The fragment length of cfDNA was determined by using Agilent 2100 Bioanalyzer. The copy numbers of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) were determined by using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) with three pairs of mitochondrial ND1 and nuclear GAPDH primers, respectively. There were short (~60 bp), medium (~167 bp), and long (>250 bp) cfDNA fragment length distributions totally obtained from CSF and serum using Agilent 2100 Bioanalyzer. The results of both qPCR and ddPCR confirmed the existence of these three cfDNA fragment ranges in CSF and serum. According to qPCR, the copy numbers of long cf-mtDNA, medium, and long cf-nDNA in CSF were significantly higher than in paired serum. In CSF, only long cf-mtDNA's copy numbers were higher than long cf-nDNA. But in serum, the copy numbers of medium and long cf-mtDNA were higher than the corresponding cf-nDNA. The cf-nDNA and cf-mtDNA with different fragment lengths differentially distributed in the CSF and serum of patients with brain disorders, which might serve as a biomarker of human brain diseases.

Chen A ，Li J ，Wang L ，Huang Q ，Zhu J ，Wen S ，Lyu J ，Wu W ... -  《-》

Very Short Mitochondrial DNA Fragments and Heteroplasmy in Human Plasma.

Zhang R ，Nakahira K ，Guo X ，Choi AM ，Gu Z ... -  《-》

Comparison of the structures and topologies of plasma extracted circulating nuclear and mitochondrial cell-free DNA.

Introduction: The function, origin and structural features of circulating nuclear DNA (cir-nDNA) and mitochondrial DNA (cir-mtDNA) are poorly known, even though they have been investigated in numerous clinical studies, and are involved in a number of routine clinical applications. Based on our previous report disproving the conventional plasma isolation used for cirDNA analysis, this work enables a direct topological comparison of the circulating structures associated with nuclear DNA and mitochondrial cell-free DNA. Materials and methods: We used a Q-PCR and low-pass whole genome sequencing (LP-WGS) combination approach of cir-nDNA and cir-mtDNA, extracted using a procedure that eliminates platelet activation during the plasma isolation process to prevent mitochondria release in the extracellular milieu. Various physical procedures, such as filtration and differential centrifugation, were employed to infer their circulating structures. Results: DSP-S cir-mtDNA mean size profiles distributed on a slightly shorter range than SSP-S. SSP-S detected 40-fold more low-sized cir-mtDNA fragments (<90 bp/nt) and three-fold less long-sized fragments (>200 bp/nt) than DSP-S. The ratio of the fragment number below 90 bp over the fragment number above 200 bp was very homogenous among both DSP-S and SSP-S profiles, being 134-fold lower with DSP-S than with SSP-S. Cir-mtDNA and cir-nDNA DSP-S and SSP-S mean size profiles of healthy individuals ranged in different intervals with periodic sub-peaks only detectable with cir-nDNA. The very low amount of cir-mtDNA fragments of short size observed suggested that most of the cir-mtDNA is poorly fragmented and appearing longer than ∼1,000 bp, the readout limit of this LP-WGS method. Data suggested that cir-nDNA is, among DNA extracted in plasma, associated with ∼8.6% of large structures (apoptotic bodies, large extracellular vesicles (EVs), cell debris…), ∼27.7% in chromatin and small EVs and ∼63.7% mainly in oligo- and mono-nucleosomes. By contrast, cir-mtDNA appeared to be preponderantly (75.7%) associated with extracellular mitochondria, either in its free form or with large EVs; to a lesser extent, it was also associated with other structures: small EVs (∼18.4%), and exosomes or protein complexes (∼5.9%). Conclusion: This is the first study to directly compare the structural features of cir-nDNA and cir-mtDNA. The significant differences revealed between both are due to the DNA topological structure contained in the nucleus (chromatin) and in the mitochondria (plasmid) that determine their biological stability in blood. Although cir-nDNA and cir-mtDNA are principally associated with mono-nucleosomes and cell-free mitochondria, our study highlights the diversity of the circulating structures associated with cell-free DNA. They consequently have different pharmacokinetics as well as physiological functions. Thus, any accurate evaluation of their biological or diagnostic individual properties must relies on appropriate pre-analytics, and optimally on the isolation or enrichment of one category of their cirDNA associated structures.

Pisareva E ，Roch B ，Sanchez C ，Pastor B ，Mirandola A ，Diab-Assaf M ，Mazard T ，Prévostel C ，Al Amir Dache Z ，Thierry AR ... -  《Frontiers in Genetics》

Urinary cell-free mitochondrial and nuclear deoxyribonucleic acid correlates with the prognosis of chronic kidney diseases.

Chang CC ，Chiu PF ，Wu CL ，Kuo CL ，Huang CS ，Liu CS ，Huang CH ... -  《-》