Roy Stuart J, Conn Simon J, Mayo Gwenda M, Athman Asmini, Gilliham Matthew
Australian Centre for Plant Functional Genomics and School of Agriculture, Food and Wine & Waite Research Institute, Glen Osmond, SA, Australia.
Methods Mol Biol. 2012;913:335-50. doi: 10.1007/978-1-61779-986-0_22.
Interrogating the cell-specific transcriptome forms an important component of understanding the role that specific cells play in assisting a plant to overcome abiotic stress. Among the challenges arising when extracting RNA from individual plant cells are: the isolation of pure cell populations; the small yield of material when isolating specific cell types, and ensuring an accurate representation of the transcriptome from each cell type after amplification of RNA. Here we describe two approaches for isolating RNA from specific cell types-single cell sampling and analysis (SiCSA) and laser capture microdissection. Isolated RNA can then be directly sampled qualitatively using reverse transcription PCR (RT-PCR) or amplified for profiling -multiple specific genes using quantitative RT-PCR and genome-wide transcript analyses.
探究细胞特异性转录组是理解特定细胞在帮助植物克服非生物胁迫中所起作用的重要组成部分。从单个植物细胞中提取RNA时面临的挑战包括：分离纯细胞群体；分离特定细胞类型时材料产量低，以及在RNA扩增后确保每种细胞类型转录组的准确表征。在这里，我们描述了两种从特定细胞类型中分离RNA的方法——单细胞采样与分析（SiCSA）和激光捕获显微切割。然后，可以使用逆转录PCR（RT-PCR）对分离的RNA进行直接定性采样，或使用定量RT-PCR和全基因组转录分析对多个特定基因进行扩增以进行分析。
