Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling
Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling
复制标题
DOI：
10.1038/nprot.2016.146
复制
发表时间：
2016-12-01
期刊：
影响因子：
14.8
通讯作者：
Rose, Jocelyn K. C.
中科院分区：
文献类型：
--
作者：
Martin, Laetitia B. B.;Nicolas, Philippe;Rose, Jocelyn K. C.
该方案能够使用激光显微切割（LM）对从番茄中分离的特定细胞或组织类型进行转录组分析。为了制备用于LM的组织，首先将水果样品固定在最佳切割温度（OCT）培养基中并在模具中冷冻。然后在使用LM仪器进行解剖之前，使用低温恒温器对组织进行切片。将收获的细胞中所含的RNA纯化并进行两轮扩增以产生足够量的RNA以产生cDNA文库。与用于分离特定细胞类型的其他几种技术不同，LM具有易于应用于任何植物物种而不必产生转基因植物的优点。使用本文所述的方案，两个样品的LM介导的细胞类型转录组学分析从组织收获到RNA测序（RNA-seq）需要近似8天，而每个额外的样品（多达总共12个样品）需要近似1天的额外LM步骤。使用这种方法获得的RNA已成功用于深度覆盖转录组分析，这是一种用于鉴定细胞或组织类型之间差异表达的基因的特别有效的策略。
This protocol enables transcriptome profiling of specific cell or tissue types that are isolated from tomato using laser microdissection (LM). To prepare tissue for LM, fruit samples are first fixed in optimal cutting temperature (OCT) medium and frozen in molds. The tissue is then sectioned using a cryostat before being dissected using an LM instrument. The RNAs contained in the harvested cells are purified and subjected to two rounds of amplification to yield sufficient quantities of RNA to generate cDNA libraries. Unlike several other techniques that are used to isolate specific cell types, LM has the advantage of being readily applied to any plant species without having to generate transgenic plants. Using the protocols described here, LM-mediated cell-type transcriptomic analysis of two samples requires similar to 8 d from tissue harvest to RNA sequencing (RNA-seq), whereas each additional sample, up to a total of 12 samples, requires similar to 1 additional day for the LM step. RNA obtained using this method has been successfully used for deep-coverage transcriptome profiling, which is a particularly effective strategy for identifying genes that are differentially expressed between cell or tissue types.