It is known that DNA content can vary in mitochondria from different cells or tissues, depending, for instance, on energy requirements. Thus, in samples from distinct areas of a specific organ, one could expect discrepancies in the ability to amplify the mtDNA based not on different levels of lesions within the sample but simply from fluctuation in the number of copies of the mitochondrial genome present. Therefore, to normalize for mitochondrial copy number, we routinely amplify an additional short fragment (no longer than 300 bp) of the mitochondrial gene under study. The idea is that the amplification of the short fragment reflects only undamaged DNA due to the low probability of introducing lesions in small segments. The results obtained with the short sequence are used to monitor the copy number of the mitochondrial genome and, more importantly, to normalize the data obtained with the large (7–15 kb) fragment. As noted above if DNA is extracted using an automated system employing the QIAcube (QIAGEN, catalog number 9001292) with the QIAamp DNA mini kit for human samples (QIAGEN, catalog number 51304), linearization of the mtDNA is necessary to get an accurate level of mtDNA. We have found that HaeII is compatible with our primer sets for mouse mtDNA (seeFig. 3) and PvuII or ClaI are compatible with our primer sets for human mtDNA.