Abstract
This chapter discusses the detection and analysis of mitochondrial DNA and RNA in muscle by in situ hybridization and single-fiber polymerase chain reaction (PCR). The power of in situ hybridization (ISH) in providing spatial information of gene location and expression is unique. ISH provides a wealth of information on the intercellular distribution of heteroplasmic mitochondrial DNA (mtDNA) populations, with its usefulness particularly evident in the study of human pathologies. The technique has been used extensively to correlate mitochondrial abnormalities with the presence of mutated mtDNAs—an analysis that provides strong support for a pathogenetic role of a specific mitochondrial genotype. Despite its qualitative nature, ISH can be used for semiquantitative determinations, if certain precautions are taken. This approach has been used to determine the ratio of rRNA and heavy-strand-encoded mRNAs in muscle of patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). Although ISH utilizing RNA probes are more widely used in typical cell and molecular biology applications, most of the experience derives from the use of DNA probes. The use of RNA probes—although requiring special care—may yield better signal-to-background results when the levels of the target sequence are low.