FISHing for RNAs: classical to single molecule approaches

Course description

Tight spatio-temporal regulation of the number and localisation of RNA molecules is key to ensuring precise control of gene expression. State-of-the-art in situ hybridisation (ISH) technologies enable visualisation and quantification of RNA molecules in a biological context, from organism and tissue to cell. Moreover, with sequential ISH technologies (e.g. MER-FISH), the RNA signature of individual cells – or even their sub-compartments – can be identified and characterised in situ. During this course, the participants will visualise subcellular localisation of selected transcripts through conventional ISH and more sensitive, quantitative and resolutive smFISH (single molecule fluorescent ISH) and HCR-ISH (hybridisation chain reaction)–based RNA-ISH hybridisation methods. Additionally, the participants will learn to design and produce their own FISH probes cost-efficiently. A particular emphasis will be on biodata (image) processing and quantitative analysis.

Audience

This course is targeted to early stage scientists (PhD and postdocs) across the field of molecular biology with an interest in application of these techniques for their research.

Modules

The course is divided into three modules containing selective content from our EMBO practical course FISHing for RNAs: classical to single molecule approaches.

• Module 1 - Dye conjugation of amino-11-ddUTP
• Module 2a and 2b - smFISH and HCR
  • Probe design for smFISH 
  • Probe labelling for smFISH
  • Assessment of smFISH probe
  • Probe design for HCR
  • Generic advices on smFISH and HCR workflow
• Module 3 - Imaging and image analysis with exercise

EMBL eCampus learning platform

These course materials are available on our EMBL eCampus learning platform. Please note that the platform is freely accessible and free of charge. After clicking the "Enter course" button, you will be redirected to the EMBL eCampus website. If you are a first-time user, you will need to create an account. If you are already an EMBL eCampus user, you can access the course directly after logging in.