Genomic tip and genomic dna buffer set kit

Quantitative PCR (QPCR) was used to assay _mtDNA damage as described previously (51 ). Briefly, total genomic DNA was isolated using QIAGEN Genomic Tip and Genomic DNA Buffer Set Kit (QIAGEN, Valencia, CA). Purified genomic DNA was quantified fluorometrically using Pico Green dsDNA reagent (Molecular Probes, Life Technologies, USA). Lambda (λ)/HindDIII DNA (Gibco Invitrogen, Paisley, UK) was used to generate a standard curve and adjust the final DNA concentration to 3 ng/μL. The “hot start” PCR used the Gene Amp XL PCR Kit (Applied Biosystems, Foster City, CA, USA) with 15 ng DNA, 1X buffer, 100 ng/μL BSA, 200 μM dNTPs, 20 pmol of each primer (Include a Table), 1.3 mM Mg²⁺ and H₂O to 45 μL. The reaction was brought to 75°C before adding 1 U/reaction enzyme (0.5 μL of polymerase in 4.5 μL H₂O). Specific primers were used to amplify a large fragment of _mtDNA (8.9 kb) to determine _mtDNA integrity; and a small fragment (139 bp) of the mitochondrial genome to monitor changes in _mtDNA copy number and to normalize the data obtained when amplifying the 8.9-kb fragment. Relative amplifications were calculated to compare KO hearts to WT hearts; these values were used to estimate quantitatively the number of lesions present in DNA, assuming a Poisson distribution as previously described (51 ).