Genome-Wide Analysis of RNA Extracted from Isolated Mitochondria

Isolating mitochondria by subcellular fractionation is a well-established method for retrieving intact and functional mitochondria. This procedure has been used to identify proteins of the mitochondria and to explore import mechanisms. Using the same method, it was shown that mitochondria can be purified along with cytoplasmic ribosomes and nuclear-encoded mRNAs attached to the outer membrane. Combining this procedure with DNA microarray analysis allows for global identification of the mRNAs associated with mitochondria, and hence a better understanding of the underlying molecular mechanisms. In this chapter, we will describe a procedure for the isolation of mitochondria from yeast and RNA purification. We will then describe the process of labeling and hybridization to DNA microarrays, and comment on a few aspects of the data analysis.