Quantifying Nuclear and Mitochondrial DNA Damage

Nuclear and mtDNA damage were assessed as described previously [9 (link),12 (link),21 (link),42 (link)]. Genomic DNA from paraffin-embedded lungs or cultured cells were extracted using the Qiagen Genomic Tip 20/G and Qiagen DNA Buffer Set (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Ex-Taq (Takara, Mountain View, CA, USA) was used for PCR with specific primers to amplify a mitochondrial gene, both in short and long-forms and also nuclear beta globin DNA [9 (link),12 (link),42 (link)]. We used PicoGreen for DNA quantification (Thermo-Fisher/Invitrogen, Waltham, MA, USA) using the FL600 microplate fluorescence reader (Thermo-Fisher, Pittsburgh, PA, USA), with excitation and emission wavelengths of 485 and 530 nm, respectively. Mitochondria short-fragment data were used to normalize fluorescence from the mitochondrial long fragment. The number of mitochondrial lesions was calculated by the equation: D = (1 − 2^{–(Δlong-Δshort)}) × 10,000 (bp)/size of the long fragment (bp).