Assessment of Mitochondrial Damage in Retinal Cells and Tissues Using Quantitative Polymerase Chain Reaction for Mitochondrial DNA Damage and Extracellular Flux Assay for Mitochondrial Respiration Activity
Mitochondrial dysfunction and genomic instability are associated with a number of retinal pathologies including age-related macular degeneration, diabetic retinopathy, and glaucoma. Consequences of mitochondrial dysfunction within cells include elevation of the rate of ROS production due to damage of electron transport chain proteins, mitochondrial DNA (mtDNA) damage, and loss of metabolic capacity. Here we introduce the quantitative polymerase chain reaction assay (QPCR) and extracellular flux assay (XF) as powerful techniques to study mitochondrial behavior. The QPCR technique is a gene-specific assay developed to analyze the DNA damage repair response in mitochondrial and nuclear genomes. QPCR has proved particularly valuable for the measurement of oxidative-induced mtDNA damage and kinetics of mtDNA repair. To assess the functional consequence of mitochondrial oxidative damage, real-time changes in cellular bioenergetics of cell monolayers can be measured with a Seahorse Biosciences XF24 analyzer. The advantages and limitations of these procedures will be discussed and detailed methodologies provided with particular emphasis on retinal oxidative stress.