FALCON-qPCR: a new method for the quantification of oxidative lesions in mitochondrial DNA

Figures

• 

  Figure 1.. FALCON-qPCR protocol overview. Schematic representation of the steps of the protocol. DNA extraction can be performed starting from as few as 10,000 cells or adjusted to different animal models. After Fpg digestion, a qPCR reaction is used to normalize the amount of mtDNA loaded in the LR-PCR. A final qPCR allows for the detection of the amount of mtDNA amplified during the LR-PCR and consequently for the following mtDNA damage evaluation. Image created with BioRender (https://app.biorender.com/citation/67c5721e9de063e41807733f).

• 

  Figure 2.. Specificity and optimization of FALCON-qPCR. (A). An example of the qPCR curves obtained by loading known concentrations of mtDNA is reported (top). The Ct values are plotted against the logarithm of the mtDNA amount loaded to extrapolate the standard curve (bottom). (B). Agarose gel analysis of LR-PCR product to evaluate the specificity of the primers. 10 µL of PCR reaction from HeLa (left), C. elegans (center), and MEFs (right) were loaded and separated on 0.8% agarose gel. (C). Agarose gel analysis of qPCR product to evaluate the specificity of the primers. 15 µL of PCR reaction from HeLa (left), C. elegans (center), and MEFs (right) were loaded and separated on 1.2% agarose gel. (D). Graph showing the relative amplification of Sample A and half the amount of the same sample (A/2) which were processed in parallel to confirm no loss of material during the steps of the protocol. The initial 1:2 ratio was maintained until the end of the procedure (** p ≤ 0.01; n = 3). (E). A total of 10,000 cells were analyzed to evaluate the amount of mtDNA damage after incubation with 200 µM H2O2 for 15 min. The protocol was performed on cells soon after the treatment (Fresh) or after freezing…

• 

  Figure 3.. FALCON-qPCR comparison with reference protocol. (A). Comparison of FALCON-qPCR with the standard protocol (Furda et al.). HeLa cells were treated with 25 μM AMA for 30 minutes in media without FBS to induce mitochondrial oxidative stress. 2 million cells were processed following Furda et al. recommendations, while 10,000 cells were used to measure mtDNA oxidative base lesions with the FALCON-qPCR. The mtDNA oxidative damage levels were quantified relative to untreated samples (CTRL) (** p ≤ 0.01; n = 3). (B). Surgical resections and needle biopsies from eight patients affected by HCC were collected from the tumoral mass and from a distal non-tumorigenic part of the liver and processed according to Furda et al. protocol or the FALCON-qPCR (n = 3). (C). Relative amount of mtDNA oxidative damage levels in HeLa cells treated with either H2O2 (left) or rotenone (right) for 15 minutes at the concentrations reported in the graphs. mtDNA oxidative damage was quantified relative to untreated cells (CTRL) (*** p ≤ 0.001; * p ≤ 0.05; n.s.: not significant; n = 3). (D). Relative amount of mtDNA oxidative damage levels in HeLa cells treated with 25 μM AMA for 30, 60 and 90 minutes. mtDNA…

• 

  Figure 4.. Assessment of mtDNA damage detection in different study models. (A-D). Acute myeloid leukemia (OCI-AML2), human glioblastoma (SF767), hepatocellular carcinoma (JHH6 and Huh7) cell lines were incubated with AMA (25 μM for OCI-AML2 and SF767, 200 μM for JHH6, and 75 μM for Huh7) for 30 minutes. mtDNA oxidative damage was quantified relative to untreated cells (CTRL) (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; n = 3). (E) Mouse embryonic fibroblasts (MEFs) were treated with 25 μM AMA for 30 minutes (*** p ≤ 0.001; n = 3). (F). C. elegans were grown for 4 days at 20°C on nematode growth medium with the concentration of Paraquat reported. Total DNA was extracted from 10 worms, approximately 10,000 cells, for each condition and compared with untreated (CTRL) to detect the levels of mtDNA oxidative lesions (** p ≤ 0.01; * p ≤ 0.05; n = 3).

• 

  Figure 5.. Analysis of two ruthenium-based compounds. (A). Chemical structure of ruthenium-based compounds C4 and C10. (B). MTT assay graphs showing the 50% inhibitory concentrations (IC50) of C4 (left) and C10 (right) (n = 5). (C). U87 cells were treated for 72 hours with 0.09 μM C4 or 0.8 μM C10 and later incubated with MitoSOX to evaluate the ROS production at the confocal microscope (left). The percentage of positive cells was evaluated. DMSO-treated cells were used as a negative control. The white bar corresponds to 50 μm (**** p ≤ 0.0001). (D). Assessment of mtDNA damage detection in U87 cells treated with either C4 or C10. Cells were incubated for 72 hours with 0.09 μM C4 or 0.8 μM C10. mtDNA oxidative damage was quantified relatively to cells treated with DMSO (* p ≤ 0.05, n = 3).