Quantitative Real Time PCR (qRT‐PCR) Evaluation of Pig Mitochondrial DNA Damage

Abstract

Mitochondrial DNA (MtDNA) damage has generated much interest in recent years as a potential biomarker after ischemia/reperfusion and traumatic injury and in critical illness. MtDNA is particularly susceptible to damage due to is close proximity to the source of generation of reactive oxygen species during metabolism and its relatively low ability to repair DNA damage compared to nuclear repair mechanisms. Reduction in MtDNA damage appears to be a good indicator of a successful intervention. QRT‐PCR has been shown to be the most sensitive and accurate method to evaluate mtDNA damage requiring only nanogram amounts of DNA. While validated primer pairs for mtDNA damage for human and rodent mtDNA damage are available, comparable sets for pig have not previously been described, yet many studies in trauma and critical illness have utilized swine models. To select primer pairs for pigs, we aligned the pig mitochondrial DNA sequence to the pig genome in the UCSC Genome Browser and primers for regions not exhibiting overlap with the nuclear genome were selected with NCBI Primer3. Using the bleomycin injury model, porcine pulmonary artery endothelial cells obtained from Cell Biologics and cultivated in MCDB 131 medium were treated for 1 hr with bleomycin (10 μg/ml). Short and long sequences were tested with pig total DNA and demonstrated that one set detected specific induction of DNA lesions. The assay was able to detect 11 lesions per 10,000 base pairs. The short primer pairs also accurately predicted mitochondrial copy number DNA as corroborated for mtDNA by published results for the pig ND4 gene. We are currently employing this assay to detect mtDNA damage in a swine model of hemorrhagic shock.Support or Funding InformationFunded by US Army Medical Research Materiel Command