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Microbiology JoVE (Open Access) Citable · DOI

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

DOI: 10.3791/3448-v
What you'll learn
  • ✓ Set up automated genome-wide RNAi screening using C. elegans feeding libraries
  • ✓ Perform high-throughput phenotypic measurements of fluorescence, size, and opacity
  • ✓ Identify genes involved in anti-fungal innate immunity via quantitative analysis
Protocol

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Difficulty
advanced
Total time
~5 days
Model organism
Caenorhabditis elegans
Biosafety
BSL-1

Steps

1
Prepare 96-well plates and RNAi bacterial cultures

Prepare 96-well NGM RNAi plates and LB plates, then culture RNAi bacterial clones from feeding libraries for overnight growth.

▶ 01:33
2
Seed RNAi bacterial clones onto NGM plates

Inoculate prepared 96-well NGM RNAi plates with cultured bacterial clones to establish RNAi feeding conditions.

▶ 03:14
3
Feed worms RNAi bacteria and infect with fungal spores

Seed worms onto RNAi-seeded plates for feeding, then infect populations with Drechmerneria coniospora spores on days 3 and 4.

▶ 04:46
4
Observe plates and prepare for automated analysis

Monitor worm phenotypes on day 5 and prepare plates for high-throughput quantitative imaging of fluorescence, size, and opacity.

▶ 06:02
5
Analyze RNAi screening results quantitatively

Process automated measurements to identify genes affecting anti-fungal immunity and other phenotypic parameters across the genome-wide screen.

▶ 07:29
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