Journal: The Journal of Biological Chemistry

Article Title: Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR

doi: 10.1016/j.jbc.2022.102574

Figure Lengend Snippet: A dPCR probe can discriminate a single nucleotide change. A , simplex droplet dPCR plots for the mutant mtDNA assay ( left ) and WT mtDNA assay ( right ) using synthetic DNA input. Both assays utilized FAM-labeled probes. Individual wells are separated by vertical lines, and the synthetic DNA input for each well is noted above the droplet plot. Approximately, 20,000 partitions (droplets) are shown within each well. The fluorescent signal of each droplet is graphically displayed according to the intensity of the signal, with stronger signal appearing at a higher amplitude. The solid horizontal line indicates the threshold between negative and positive droplets. B , duplex droplet dPCR plots for the mutant mtDNA assay ( top panel ) and reference mtDNA assay ( middle panel ) using different amounts of synthetic DNA input. The mutant mtDNA assay used a FAM-labeled probe, while the reference mtDNA assay used a HEX-labeled probe. The synthetic DNA input for each well is noted above the droplet plots. Horizontal lines are drawn to differentiate the distinct droplet populations in the mutant mtDNA assay (high-amplitude positive, low-amplitude positive, and negative) and reference mtDNA assay (positive and negative). The bottom panel shows the calculated percentage of each synthetic DNA. The percentage of mutant DNA input was quantified by dividing the concentration (copies/μl) of high-amplitude FAM-positive droplets by the concentration of HEX-positive droplets. The percentage of WT DNA input was quantified by dividing the concentration of low-amplitude FAM-positive droplets by the concentration of HEX-positive droplets. C , duplex droplet dPCR plots for the WT mtDNA assay ( top panel ) and reference mtDNA assay ( middle panel ) using different amounts of synthetic DNA input. The WT mtDNA assay used a FAM-labeled probe, while the reference mtDNA assay used a HEX-labeled probe. The synthetic DNA input for each well is noted above the droplet plots. Horizontal lines are drawn to differentiate the distinct droplet populations, as described above. The bottom panel shows the calculated percentage of each synthetic DNA input. The percentage of WT DNA input was quantified by dividing the concentration (copies/μl) of high-amplitude FAM-positive droplets by the concentration of HEX-positive droplets. The percentage of mutant DNA input was quantified by dividing the concentration of low-amplitude FAM-positive droplets by the concentration of HEX-positive droplets. NTC, no template control; mtDNA, mitochondrial DNA; dPCR, digital PCR.

Article Snippet: Droplets were analyzed using a QX200 droplet reader (Bio-Rad), and QuantaSoft analysis software (Bio-Rad) was used to acquire and analyze data. mtDNA copy number was calculated by multiplying the concentration (copies/μl) of reference mtDNA–positive droplets by the dilution factor (400) and dividing by the concentration of APOC3-positive droplets.

Techniques: Mutagenesis, Labeling, Concentration Assay, Digital PCR

Journal: The Journal of Biological Chemistry

Article Title: Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR

doi: 10.1016/j.jbc.2022.102574

Figure Lengend Snippet: dPCR can be used to quantify heteroplasmy in cellular DNA samples . A , duplex droplet dPCR plots for the mutant mtDNA assay ( top ) and reference mtDNA assay ( bottom ) using various m.3243A > G heteroplasmic cell lines (clones #1–14). The mutant mtDNA assay used a FAM-labeled probe, while the reference mtDNA assay used a HEX-labeled probe. Horizontal lines are drawn to differentiate the distinct droplet populations in the mutant mtDNA assay (high-amplitude positive, low-amplitude positive, and negative) and reference mtDNA assay (positive and negative). The calculated percentage of mutant mtDNA is displayed above each sample. B , heteroplasmy quantification of various m.3243A > G heteroplasmic cell lines using the duplex of the mutant mtDNA assay and reference mtDNA assay shown in Fig. 3 A . The percentage of mutant mtDNA was quantified by dividing the concentration (copies/μl) of high-amplitude FAM-positive droplets by the concentration of HEX-positive droplets. The percentage of WT mtDNA was quantified by dividing the concentration of low-amplitude FAM-positive droplets by the concentration of HEX-positive droplets. C , correlation analysis comparing the three standard methods for heteroplasmy quantification (Sanger sequencing, Psp OMI ”Last cycle hot” PCR/RFLP, and Hae III ”Last cycle hot” PCR/RFLP) to dPCR. The R value comparing dPCR to Sanger sequencing was 0.9938, dPCR to Psp OMI ”Last cycle hot” PCR/RFLP was 0.9924, and dPCR to Hae III ”Last cycle hot” PCR/RFLP was 0.9954. mtDNA, mitochondrial DNA; dPCR, digital PCR; RFLP, restriction fragment-length polymorphism.

Article Snippet: Droplets were analyzed using a QX200 droplet reader (Bio-Rad), and QuantaSoft analysis software (Bio-Rad) was used to acquire and analyze data. mtDNA copy number was calculated by multiplying the concentration (copies/μl) of reference mtDNA–positive droplets by the dilution factor (400) and dividing by the concentration of APOC3-positive droplets.

Techniques: Mutagenesis, Clone Assay, Labeling, Concentration Assay, Sequencing, Digital PCR

Journal: The Journal of Biological Chemistry

Article Title: Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR

doi: 10.1016/j.jbc.2022.102574

Figure Lengend Snippet: 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.

Article Snippet: Droplets were analyzed using a QX200 droplet reader (Bio-Rad), and QuantaSoft analysis software (Bio-Rad) was used to acquire and analyze data. mtDNA copy number was calculated by multiplying the concentration (copies/μl) of reference mtDNA–positive droplets by the dilution factor (400) and dividing by the concentration of APOC3-positive droplets.

Techniques: Isolation, Labeling, Concentration Assay, Digital PCR

Journal: The Journal of Biological Chemistry

Article Title: Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR

doi: 10.1016/j.jbc.2022.102574

Figure Lengend Snippet: dPCR can be used to simultaneously quantify heteroplasmy and mtDNA copy number. A , sample duplex droplet dPCR plots for the mutant mtDNA assay ( top ) and 18S rDNA nuclear assay ( bottom ) using cellular DNA isolated from clone #2. 0.225 ng of DNA was used in the duplex. The mutant mtDNA assay used a FAM-labeled probe, while the 18S rDNA nuclear assay used a HEX-labeled probe. Horizontal lines are drawn to differentiate the distinct droplet populations in the mutant mtDNA assay (high-amplitude positive, low-amplitude positive, and negative) and 18S rDNA nuclear assay (positive and negative). B , quantification of mtDNA copy number using duplex dPCR. mtDNA copy number was calculated by dividing the concentration (copies/μl) of both positive droplet populations (high-amplitude + low-amplitude) in the mutant mtDNA assay by the concentration of positive droplets for the 18S rDNA nuclear assay. C , quantification of mtDNA heteroplasmy using the mutant mtDNA assay. The percentage of mutant mtDNA was quantified by dividing the concentration (copies/μl) of high-amplitude FAM-positive droplets by the concentration of total FAM-positive droplets (high-amplitude + low-amplitude). The percentage of WT mtDNA was quantified by dividing the concentration (copies/μl) of low-amplitude FAM-positive droplets by the concentration of total FAM-positive droplets (high-amplitude + low-amplitude). mtDNA, mitochondrial DNA; dPCR, digital PCR.

Article Snippet: Droplets were analyzed using a QX200 droplet reader (Bio-Rad), and QuantaSoft analysis software (Bio-Rad) was used to acquire and analyze data. mtDNA copy number was calculated by multiplying the concentration (copies/μl) of reference mtDNA–positive droplets by the dilution factor (400) and dividing by the concentration of APOC3-positive droplets.

Techniques: Mutagenesis, Isolation, Labeling, Concentration Assay, Digital PCR

Journal: The Journal of Biological Chemistry

Article Title: Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR

doi: 10.1016/j.jbc.2022.102574

Figure Lengend Snippet: General applicability of the dPCR approach for heteroplasmic mtDNA mutations. dPCR was used to determine mtDNA heteroplasmy and mtDNA copy number using DNA samples isolated from heteroplasmic mice carrying the m.5024C > T mutation. DNA was isolated from the tibialis anterior (sample #1, A ) and cortex (sample #2, B ). Two different dPCR assays were used: one specific to the WT m.5024C and one in the mouse 18S rDNA gene. The WT assay used a FAM-labeled probe, while the 18S rDNA nuclear assay used a Cy5-labeled probe. Horizontal lines are drawn to differentiate the distinct droplet populations in the WT mtDNA assay (high-amplitude positive, low-amplitude positive, and negative) and 18S rDNA nuclear assay (positive and negative). The percentage of WT mtDNA was quantified by dividing the concentration (copies/μl) of high-amplitude FAM-positive droplets by the concentration of total FAM-positive droplets (high-amplitude + low-amplitude). The percentage of mutant mtDNA was quantified by dividing the concentration of low-amplitude FAM-positive droplets by the concentration of total FAM-positive droplets (high-amplitude + low-amplitude). mtDNA copy number was calculated by dividing the concentration of both positive droplet populations (high-amplitude + low-amplitude) in the WT mtDNA assay by the concentration of positive droplets for the 18S rDNA nuclear assay. The same samples used to detect the levels of heteroplasmy by dPCR were also analyzed by RFLP, as indicated on the right . mtDNA, mitochondrial DNA; dPCR, digital PCR; RFLP, restriction fragment-length polymorphism.

Article Snippet: Droplets were analyzed using a QX200 droplet reader (Bio-Rad), and QuantaSoft analysis software (Bio-Rad) was used to acquire and analyze data. mtDNA copy number was calculated by multiplying the concentration (copies/μl) of reference mtDNA–positive droplets by the dilution factor (400) and dividing by the concentration of APOC3-positive droplets.

Techniques: Isolation, Mutagenesis, Labeling, Concentration Assay, Digital PCR