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. 2022 Oct 6;298(11):102574. doi: 10.1016/j.jbc.2022.102574

Figure 4.

Figure 4

dPCR can be used to quantify mtDNA copy number. A, sample simplex droplet dPCR plots for the reference mtDNA assay (top) and APOC3 nuclear assay (bottom) using different amounts of cellular DNA isolated from clone #2. Both assays utilized HEX-labeled probes. The solid horizontal line indicates the threshold between negative and positive droplets for each assay. B, quantification of mtDNA copy number using simplex dPCR. 0.225 ng of DNA was used in the reference mtDNA assay, while 90 ng of DNA was used in the APOC3 nuclear assay. The resulting concentration (copies/μl) of positive droplets for the reference mtDNA assay was multiplied by the DNA dilution factor (400) and then divided by the concentration of positive droplets for the APOC3 nuclear assay to yield mtDNA copy number per copy of APOC3. C, sample duplex droplet dPCR plots for the reference mtDNA assay (top) and 18S rDNA nuclear assay (bottom) using cellular DNA isolated from clone #2. The reference mtDNA assay used a HEX-labeled probe, while the 18S rDNA nuclear assay used a FAM-labeled probe. The solid horizontal line indicates the threshold between negative and positive droplets for each assay. D, quantification of mtDNA copy number using duplex dPCR. 0.225 ng of DNA was used in the duplex containing the reference mtDNA and 18S rDNA nuclear assays. The resulting concentration (copies/μl) of positive droplets for the reference mtDNA assay was divided by the concentration of positive droplets for the 18S rDNA nuclear assay to yield mtDNA copy number per copy of 18S rDNA. E, comparison of mtDNA copy number by dPCR simplex, dPCR duplex, and qPCR. qPCR copy number was calculated using either MT-ND1 or MT-CO1 as the mtDNA reference gene and ACTIN as the nDNA reference gene. The calculated copy numbers were normalized to the first replicate at the first timepoint. F, correlation analysis of simplex dPCR and duplex dPCR for determining mtDNA copy number. The R value was 0.7990. mtDNA, mitochondrial DNA; dPCR, digital PCR; nDNA, nuclear DNA.