Thiel Johannes, Weier Diana, Weschke Winfriede
Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany.
Methods Mol Biol. 2011;755:461-75. doi: 10.1007/978-1-61779-163-5_39.
Laser microdissection provides a useful method for isolating specific cell types from complex biological samples for downstream applications. In contrast to the texture of mammalian cells, most plant tissues exhibit a cell organization with hard, cellulose-containing cell walls, large vacuoles, and air spaces, thus complicating tissue preparation and extraction of macromolecules such as DNA and RNA. Especially, barley seeds show cell types with enormous differences in osmolarity (degenerating and differentiating tissues) and contain high amounts of the main storage product starch, thus requiring specific procedures for morphological preservation and RNA extraction. In this study, we report about methods allowing tissue-specific gene expression profiling of developing barley seeds. Details on aspects of tissue preparation, including fixation and embedding procedures, laser-capture microdissection, RNA isolation, and linear mRNA amplification to produce high-quality labelled probes for large-scale expression analysis are provided. Particular emphasis is placed on the fidelity of transcript data obtained by the developed methods in relation to the in vivo transcriptome.
激光显微切割提供了一种从复杂生物样本中分离特定细胞类型以用于下游应用的有用方法。与哺乳动物细胞的质地不同，大多数植物组织呈现出一种细胞组织结构，具有坚硬的、含纤维素的细胞壁、大液泡和细胞间隙，因此使得组织制备以及DNA和RNA等大分子的提取变得复杂。特别是，大麦种子显示出渗透压差异极大的细胞类型（退化和分化组织），并且含有大量主要的储存产物淀粉，因此需要特定的形态保存和RNA提取程序。在本研究中，我们报告了允许对发育中的大麦种子进行组织特异性基因表达谱分析的方法。提供了关于组织制备方面的详细信息，包括固定和包埋程序、激光捕获显微切割、RNA分离以及线性mRNA扩增，以产生用于大规模表达分析的高质量标记探针。特别强调了所开发方法获得的转录本数据相对于体内转录组的保真度。
