RNA fluorescence in situ hybridization for high-content screening

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Single molecule RNA imaging using fluorescent in situ hybridization (FISH) offers quantitative mRNA abundance and localization data within single cells.
• Growing interest exists in identifying genetic or chemical modifiers affecting RNA abundance and localization.
• Microsatellite expansion in RNA can cause toxic gain-of-function through mislocalization and RNA-binding protein sequestration.

Purpose of the Study:

• To describe detailed methods for performing single molecule RNA FISH in multiwell plates.
• To adapt protocols for high-content screening (HCS) microscopy.
• To provide recommendations for successful HCS microscopy with high magnification objectives.

Main Methods:

• Detailed protocols for single molecule RNA FISH in multiwell plates.
• Adaptation of standard RNA FISH with fluorescent oligonucleotide probes for HCS.
• Adaptation of single molecule inexpensive FISH (smiFISH) for HCS.

Main Results:

• Established protocols for high-throughput RNA FISH in multiwell plates.
• Demonstrated applicability of both standard RNA FISH and smiFISH for HCS.
• Provided guidelines for optimizing HCS microscopy with high magnification objectives.

Conclusions:

• Single molecule RNA FISH is a viable method for high-content screening of RNA localization modifiers.
• The described protocols facilitate large-scale screening for therapeutic targets.
• Optimized HCS microscopy protocols enhance the utility of RNA FISH for biological discovery.