粟酒裂殖酵母线粒体及线粒体RNA的提取

The Extraction of Schizosaccharomyces pombe Mitochondria and Mitochondrial RNA

粟酒裂殖酵母是一个研究线粒体生物合成的优秀的真核生物模型。本研究利用差速和蔗糖梯度离心法获得了粟酒裂殖酵母线粒体。用BCA法检测了线粒体蛋白浓度,用Western-blot和电子显微镜检测了线粒体纯度以及完整度。以纯化获得的线粒体进行RNAs提取,得到包括t RNA和小RNA在内的线粒体RNA,并用电泳和定量PCR检测了线粒体RNAs的纯度和完整度。结果表明,我们得到了高质量的粟酒裂殖酵母线粒体和线粒体RNAs。线粒体和线粒体RNA制备方法为研究蛋白靶标、蛋白定位和线粒体RNA加工提供了技术工具。

The fission yeast Schizosaccharomyces pombe has served as a excellent model eukaryote to investigate mitochondrial biogenesis. In this study, the mitochondrial of S. pombe was purified by differential centrifugation and sucrose density gradient centrifugation. And then the protein concentration, the purity and integrity of the mitochondrial preparation was determined by BCA assay, western-blot analysis and electron microscopy, respectively. Subsequently, RNAs including tRNAs and small RNAs were harvested from isolated mitochondria, and the quantity and purity of the harvested RNAs were validated by electrophoresis and qRT-PCR. These results showed that the high quality mitochondria and mitochondrial RNAs of S. pombe were obtained, which suggested that this preparation approach would provide a technique tool for investigating protein targeting, localization, and mitochondrial RNA processing in mitochondrial and mitochondrial RNA.