Cagnone 2015 Methods Mol Biol
|Cagnone G, Vaghjiani V, Lee W, Sun C, Johnson J, Yeung KY, St John JC (2015) Analysis of the mitochondrial DNA and its replicative capacity in induced pluripotent stem cells. Methods Mol Biol 1357:231-67.|
Abstract: The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis.
• Keywords: Mitochondrial DNA, Variants, Next-generation sequencing, Pluripotent cells, Differentiation, Transcription and replication, Oxygen consumption, ATP
Labels: MiParea: Respiration, Instruments;methods, mtDNA;mt-genetics
Organism: Human Tissue;cell: Stem cells Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ET Pathway: ROX HRR: Oxygraph-2k