Quantitative and automated high-throughput genome-wide RNAi screens in C. elegans

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• RNA interference (RNAi) is crucial for understanding gene function, especially at the whole-genome scale.
• Existing RNAi screening methods in C. elegans can be laborious, limiting throughput.
• Semi-automated protocols have improved efficiency but often rely on imaging and image analysis.

Purpose of the Study:

• To develop and validate an alternative high-throughput protocol for genome-wide RNAi screening in C. elegans.
• To enable screens on solid medium, facilitating studies like host-pathogen interactions.
• To improve data management and sample tracking for increased efficiency.

Main Methods:

• Utilized robotic handling of bacterial RNAi clones for automated screening.
• Employed quantitative analysis with the COPAS Biosort system.
• Integrated the MBioLIMS software for enhanced data management and sample tracking.
• Conducted screens on solid medium plates.

Main Results:

• The protocol allows for high-throughput screening, with over 3,000 RNAi clones tested and analyzed per week.
• Demonstrated the method's utility in quantifying gene importance for anti-fungal innate immunity in C. elegans.
• Successfully used a transgenic strain with dual fluorescent reporters for quantitative assessment of gene function.

Conclusions:

• The described protocol significantly increases throughput for genome-wide RNAi screens in C. elegans.
• This method facilitates studies requiring solid medium, such as host-pathogen interactions.
• The entire C. elegans genome can be screened in under two months using this approach.