Document Detail


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.
MedLine Citation:
PMID:  23150372     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Stuart G Jarrett; Bärbel Rohrer; Nathan R Perron; Craig Beeson; Michael E Boulton
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  935     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2013  
Date Detail:
Created Date:  2012-11-15     Completed Date:  2013-04-17     Revised Date:  2013-04-26    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  227-43     Citation Subset:  IM    
Affiliation:
Department of Molecular and Biomedical Pharmacology, College of Medicine, University of Kentucky, Lexington, KY, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Culture Techniques / methods
DNA Damage*
DNA, Mitochondrial / genetics*,  isolation & purification
Humans
Mitochondria / genetics*,  metabolism*,  pathology
Oxidative Stress
Oxygen / metabolism
Real-Time Polymerase Chain Reaction / methods*
Retina / cytology*,  metabolism,  pathology
Grant Support
ID/Acronym/Agency:
EY019688/EY/NEI NIH HHS; EY021626/EY/NEI NIH HHS; R01 EY019320/EY/NEI NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Mitochondrial; 7782-44-7/Oxygen

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