Safieddine Adham, Coleno Emeline, Lionneton Frederic, Traboulsi Abdel-Meneem, Salloum Soha, Lecellier Charles-Henri, Gostan Thierry, Georget Virginie, Hassen-Khodja Cédric, Imbert Arthur, Mueller Florian, Walter Thomas, Peter Marion, Bertrand Edouard
Institut de Génétique Moléculaire de Montpellier, University of Montpellier, CNRS, Montpellier, France.
Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, Laboratoire de Biologie du Développement, Paris, France.
Nat Protoc. 2023 Jan;18(1):157-187. doi: 10.1038/s41596-022-00750-2. Epub 2022 Oct 24.
The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity.
通过单分子荧光原位杂交（smFISH）在天然亚细胞环境中可视化RNA的能力，重塑了我们对基因表达和细胞功能的理解。smFISH的一个主要障碍是难以以中高通量形式进行系统实验，主要原因是生成单个荧光探针集的成本很高。在这里，我们展示了高通量smFISH（HT-smFISH），这是一种简单且经济高效的方法，用于在96孔板中对数百至数千个单个内源性RNA分子进行成像。HT-smFISH使用从大量未标记寡核苷酸体外转录的RNA探针。这使得能够为许多RNA种类生成单个探针，取代商业DNA探针集。因此，与许多smFISH方法相比，HT-smFISH降低了每个目标RNA的成本，并且在设计上易于扩展且灵活。我们提供了一个方案，该方案结合了寡核苷酸库设计、探针集生成、优化的杂交条件以及图像采集和分析指南。该流程需要标准分子生物学工具、细胞培养和荧光显微镜的知识。大约20天即可完成。简而言之，HT-smFISH专为以单分子灵敏度对RNA进行成像的中高通量筛选而设计。
