Mitochondrial DNA (mtDNA) biogenesis: visualization and duel incorporation of
BrdU and EdU into newly synthesized mtDNA in vitro.
Authors Lentz SI, Edwards JL, Backus C, McLean LL, Haines KM, Feldman EL
Submitted By Eva Feldman on 7/10/2013
Status Published
Journal The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
Year 2010
Date Published 2/1/2010
Volume : Pages 58 : 207 - 218
PubMed Reference 19875847
Abstract Mitochondria are key regulators of cellular energy and are the focus of a large
number of studies examining the regulation of mitochondrial dynamics and
biogenesis in healthy and diseased conditions. One approach to monitoring
mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA)
replication. We developed a sensitive technique to visualize newly synthesized
mtDNA in individual cells to study mtDNA replication within subcellular
compartments of neurons. The technique combines the incorporation of
5-bromo-2-deoxyuridine (BrdU) and/or 5-ethynyl-2'-deoxyuridine (EdU) into mtDNA,
together with a tyramide signal amplification protocol. Employing this
technique, we visualized and measured mtDNA biogenesis in individual cells. The
labeling procedure for EdU allows for more comprehensive results by allowing the
comparison of its incorporation with other intracellular markers, because it
does not require the harsh acid or enzyme digests necessary to recover the BrdU
epitope. In addition, the utilization of both BrdU and EdU permits sequential
pulse-chase experiments to follow the intracellular localization of mtDNA
replication. The ability to quantify mitochondrial biogenesis provides an
essential tool for investigating the alterations in mitochondrial dynamics
involved in the pathogenesis of multiple cellular disorders, including
neuropathies and neurodegenerative diseases.

Investigators with authorship
Eva FeldmanUniversity of Michigan