Zhang Xiaodan, Hu Chuansheng, Huang Chen, Wei Ying, Li Xiaowei, Hu Miaomiao, Li Hua, Wu Ji, Czajkowsky Daniel M, Guo Yan, Shao Zhifeng
State Key Laboratory of Oncogenes and Related Genes, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China.
Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Front Cell Dev Biol. 2022 Mar 25;10:853188. doi: 10.3389/fcell.2022.853188. eCollection 2022.
The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue, as local interactions precipitate intra-cellular events that often lead to changes in expression. However, our understanding of these processes in tissues, whether healthy or diseased, is limited at present owing to the difficulty in acquiring comprehensive transcriptional programs of spatially- and phenotypically-defined cells . Here we present a robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved transcriptional programs with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues sufficient to resolve even isoforms. The protocol is optimized to protect the RNA with a small molecule inhibitor, the ribonucleoside vanadyl complex (RVC), which thereby enables the typical time-consuming immunostaining and laser capture steps of this procedure during which RNA is usually severely degraded in existing approaches. The efficacy of this approach is exemplified by the characterization of differentially expressed genes between the mouse small intestine lacteal cells at the tip versus the main capillary body, including those that function in sensing and responding to local environmental cues to stimulate intra-cellular signalling. With the extensive repertoire of specific antibodies that are presently available, our method provides an unprecedented capability for the analysis of transcriptional networks and signalling pathways during development, pathogenesis, and aging of specific cell types within native tissues.
组织的功能不仅从根本上依赖于组成细胞的表型，还取决于它们在组织中的空间组织，因为局部相互作用会引发细胞内事件，这些事件常常导致表达变化。然而，由于难以获取空间和表型定义明确的细胞的全面转录程序，我们目前对健康或患病组织中这些过程的理解是有限的。在这里，我们提出了一种基于免疫荧光引导激光捕获显微切割的强大方法（免疫LCM-RNAseq），用于从速冻或经RNA Later处理的临床组织中获取精细解析的转录程序，所需细胞数量少至几十个，甚至足以解析异构体。该方案经过优化，使用小分子抑制剂核糖核苷钒配合物（RVC）保护RNA，从而能够在该过程中进行通常耗时的免疫染色和激光捕获步骤，而在现有方法中，RNA通常会在这些步骤中严重降解。这种方法的有效性通过对小鼠小肠尖端乳糜细胞与主要毛细血管体之间差异表达基因的表征得到了体现，其中包括那些在感知和响应局部环境线索以刺激细胞内信号传导中起作用的基因。凭借目前可用的大量特异性抗体，我们的方法为分析天然组织中特定细胞类型在发育、发病机制和衰老过程中的转录网络和信号通路提供了前所未有的能力。
