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

Stuart G. Jarrett, Bärbel Rohrer, Nathan R. Perron, Craig Beeson, Michael E. Boulton

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
Pages227-243
Number of pages17
Volume935
DOIs
StatePublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume935
ISSN (Print)10643745

Fingerprint

Mitochondrial DNA
DNA Damage
Respiration
DNA Repair
Polymerase Chain Reaction
Smegmamorpha
Mitochondrial Genome
Genomic Instability
Macular Degeneration
Diabetic Retinopathy
Electron Transport
Glaucoma
Energy Metabolism
Carrier Proteins
Oxidative Stress
Pathology
Genes

Keywords

  • Extracellular flux assay
  • Mitochondria
  • Photoreceptor cell, Oxidative stress
  • Quantitative polymerase chain reaction
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Jarrett, S. G., Rohrer, B., Perron, N. R., Beeson, C., & Boulton, M. E. (2013). 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. In Methods in Molecular Biology (Vol. 935, pp. 227-243). (Methods in Molecular Biology; Vol. 935). https://doi.org/10.1007/978-1-62703-080-9-16

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. / Jarrett, Stuart G.; Rohrer, Bärbel; Perron, Nathan R.; Beeson, Craig; Boulton, Michael E.

Methods in Molecular Biology. Vol. 935 2013. p. 227-243 (Methods in Molecular Biology; Vol. 935).

Research output: Chapter in Book/Report/Conference proceedingChapter

Jarrett, Stuart G. ; Rohrer, Bärbel ; Perron, Nathan R. ; Beeson, Craig ; Boulton, Michael E. / 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. Methods in Molecular Biology. Vol. 935 2013. pp. 227-243 (Methods in Molecular Biology).
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