| Added by | pcoopman |
|---|---|
| Group name | EquipePC |
| Item Type | Journal Article |
| Title | HT-smFISH: a cost-effective and flexible workflow for high-throughput single-molecule RNA imaging |
| Creator | Safieddine et al. |
| Author | Adham Safieddine |
| Author | Emeline Coleno |
| Author | Frederic Lionneton |
| Author | Abdel-Meneem Traboulsi |
| Author | Soha Salloum |
| Author | Charles-Henri Lecellier |
| Author | Thierry Gostan |
| Author | Virginie Georget |
| Author | Cédric Hassen-Khodja |
| Author | Arthur Imbert |
| Author | Florian Mueller |
| Author | Thomas Walter |
| Author | Marion Peter |
| Author | Edouard Bertrand |
| Abstract | 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. |
| Publication | Nature Protocols |
| Volume | 18 |
| Issue | 1 |
| Pages | 157-187 |
| Date | 2023-01 |
| Journal Abbr | Nat Protoc |
| Language | eng |
| DOI | 10.1038/s41596-022-00750-2 |
| ISSN | 1750-2799 |
| Short Title | HT-smFISH |
| Library Catalog | PubMed |
| Extra | PMID: 36280749 |
| Tags | Cost-Benefit Analysis, Diagnostic Imaging, In Situ Hybridization, Fluorescence, original, RNA, Workflow |
| Date Added | 2023/11/15 - 18:10:54 |
| Date Modified | 2023/11/15 - 18:11:56 |
| Notes and Attachments | PubMed entry (Attachment) Version soumise (Attachment) |
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