Eliyahu Erez, Melamed Daniel, Arava Yoav
Technion–Israel Institute of Technology, Haifa, Israel.
Methods Mol Biol. 2011;714:287-99. doi: 10.1007/978-1-61779-005-8_18.
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.
通过亚细胞分级分离来分离线粒体是一种成熟的获取完整且功能正常的线粒体的方法。该程序已被用于鉴定线粒体蛋白质并探索导入机制。使用相同的方法表明，线粒体可以与附着在外膜上的细胞质核糖体和核编码mRNA一起被纯化。将此程序与DNA微阵列分析相结合，可以全面鉴定与线粒体相关的mRNA，从而更好地理解潜在的分子机制。在本章中，我们将描述从酵母中分离线粒体和RNA纯化的程序。然后，我们将描述标记和与DNA微阵列杂交的过程，并对数据分析的几个方面进行评论。
