Robust Acquisition of Spatial Transcriptional Programs in Tissues With Immunofluorescence-Guided Laser Capture Microdissection

作者：Xiaodan Zhang, Chuansheng Hu, Chen Huang, Ying Wei, Xiaowei Li, Miaomiao Hu, Hua Li, Ji Wu, Daniel M. Czajkowsky, Yan Guo, Zhifeng Shao · 发表于：Frontiers in Cell and Developmental Biology · 年份：2022 · DOI：10.3389/fcell.2022.853188 · 被引用次数：14 · 研究领域：Molecular Biology Techniques and Applications、Single-cell and spatial transcriptomics、RNA Research and Splicing

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 in situ . 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 prov...