Table 1.
Primer sequences used for measuring heteroplasmy and mtDNA copy number.
Fig 1.
Analysis of primer efficiency.
Standard curves were generated by qPCR using as template 5-fold serial dilutions of heteroplasmic gDNA or pDNA (25, 5, 1 and 0.2 ng per reaction). First-degree linear regressions were fitted for the log of input amount of template versus the Ct (A) or the ∆Ct (B) values for serial-diluted DNAs. The ∆Ct was calculated by subtracting either CtB6 from CtNZB (heteroplasmy quantification) or CtmtDNA from CtApob (copy number quantification). Comparison of amplification efficiency between primer pairs (ARMS2/MT14 vs. ARMS22/MT20 and MT14/MT15 vs. OIMR1544/OIMR3580) was found similar as the slope values from linear regressions were equal or smaller than 0.1 (B). Moreover, the use of gDNA or pDNA for heteroplasmy quantification did not affect slope values.
Fig 2.
Specificity of the ARMS-qPCR approach for amplification of target mtDNA.
Using samples from homoplasmic mice (either NZB or B6), non-specific amplification of non-target mtDNA accounted for only 0.01% of target amplification. Analysis of samples from heteroplasmic mice showed that levels as low as 0.1% of mtDNA of NZB or B6 origin could be detected. Individual Ct values (cycle at which plots crossed the threshold) are denoted in the inset.
Fig 3.
Relationship between the input level of NZB gDNA and the level of NZB mtDNA estimated by ARMS-qPCR.
Different proportions of gDNA from NZB mice (100%, 99.9%, 99.5%, 99%, 95%, 90%, 75%, 50%, 25%, 10%, 5%, 1%, 0.5%, 0.1% and 0%) were mixed with gDNA from B6 mice to be analyzed by ARMS-qPCR. Filled circles depict the use of 0.5 ng of DNA per reaction (duplicates) whereas empty circles depict the use of 5.0 ng of DNA (triplicates). The relationship between the input level of NZB gDNA and the level of NZB mtDNA determined by ARMS-qPCR was analyzed by calculating Pearson’s correlation coefficient (r). P value is denoted in the inset.
Fig 4.
Level of NZB mtDNA in heteroplasmic mouse embryos produced by cytoplasmic transfer.
Zygotes were centrifuged prior to cytoplasmic transfer to enable production of embryos with higher levels of NZB mtDNA (filled circles). For comparison, cytoplasmic transfer was also performed without centrifugation (empty circles). Embryos from both groups were evaluated with regards to the level of NZB mtDNA at the zygote and blastocyst stages by ARMS-qPCR. Bars represent the means. P values for the effect of Group, Stage and Group x Stage are denoted in the inset.
Fig 5.
Level of NZB mtDNA across generations in a heteroplasmic mouse lineage produced by cytoplasmic transfer.
Circles depict the level of NZB mtDNA in mice estimated by ARMS-qPCR from ear biopsies. Filled circles depict females that were backcrossed (BC) with B6 males to obtain the next generation progeny (BC1, BC2, BC3, BC4 and BC5). The level of NZB mtDNA was not determined for males, except in the founder lineage where males are depicted by empty circles. In the remaining generations (BC1, BC2, BC3, BC4 and BC5) empty circles depict females that were not mated. Bars represent the means.
Fig 6.
Comparison of the level of NZB mtDNA among tissues of heteroplasmic males.
Bars depict the level of NZB mtDNA estimated by ARMS-qPCR from mice aged four months. Tissues analyzed included brain, heart, liver and tail. Values are reported as mean ± SEM. Bars with different letters denote a significant difference among tissues (P < 0.05).
Fig 7.
Comparison of mtDNA copy number among tissues of heteroplasmic males.
Bars depict mtDNA copy number per cell from mice aged four months. Tissues analyzed included brain, heart, liver and tail. Values are reported as mean ± SEM. Bars with different letters denote a significant difference among tissues (P < 0.05).