Abstract
This chapter provides an overview of microdissection and analytical polymerase chain reaction (PCR) techniques and some example protocols applicable to the study of mitochondrial DNA (mtDNA). The minute amounts of DNA recoverable from microdissected samples, typically in the pico- to nanogram range, necessitate the use of specialized PCR-based protocols for analysis. Even within the spectrum of samples obtained using microdissection, mtDNA abundance can vary considerably from that extracted from a single cell to samples derived from multiple muscle fibers. Moreover, optimizing a microdissection and mtDNA analysis protocol usually requires some degree of experimentation. An important factor to bear in mind in relation to mtDNA analysis is the potential for the occurrence of mtDNA depletion or ΔmtDNA species in tissues of interest that can also impact PCR efficiency relative to normal tissues. Analytical PCR of microdissected samples is emerging as a powerful tool for investigating the biology of mtDNA. Clearly, for complex analyses, access to specialized equipment is required, but as institutional investment in tools, such as laser microdissection microscopes and real-time PCR machines increases, access within the mitochondrial research community will surely also increase. Application of these techniques to more diverse model systems and tissue archives can only benefit the field, both in terms of the understanding of mtDNA at the cellular level and in the development of improved analytical techniques. Obtaining sufficient DNA for analysis remains the most challenging aspect of working with microdissected samples.
