We describe a protocol for performing RNA interference (RNAi) screens in C. elegans in liquid culture in 96-well plates. The procedure allows thousands of RNAi feeding experiments to be performed per investigator per day. By comparing RNAi phenotypes between wild-type worms and worms carrying a defined genetic mutation we have used this protocol to systematically identify synthetic genetic interactions between genes. We also describe how the protocol can be adapted to target two genes simultaneously by combinatorial RNAi.
C. elegans is a unique model organism for reverse genetic analysis, because RNAi can be delivered systemically by feeding worms bacteria that express dsRNA targeting any gene of interest . The availability of a genome-wide RNAi feeding library  has facilitated the completion of multiple genome-wide loss-of-function phenotypic screens (reviewed in ). However RNAi feeding experiments are normally performed using bacterial feeding on agar plates (“plate feeding”), which has a throughput that restricts the number of screens that can be performed to one or two genome-wide screens per study.
To improve the throughput of RNAi screens in C. elegans, we developed a protocol for screening in liquid culture in 96-well plates . RNAi feeding bacteria are grown overnight in 96-well plates and re-suspended in nematode growth media (NGM). Approximately 10 synchronised first larval stage (L1) worms obtained by filtration (or bleaching adults and hatching overnight) are dispensed into each well of a flat-bottom 96-well plate, to which 40 µl of bacterial suspension is added. The plates are incubated for 4 days with shaking at 20°C allowing sufficient time for the L1 worms to grow to adults, lay eggs and these eggs to hatch and develop into larvae. By this stage worms will have consumed most of their food, resulting in clearing of the bacterial suspension and allowing easy scoring of phenotypes on a dissecting microscope.
There are two main advantages to this protocol. First, all steps of the protocol use 96-well plates, allowing easy setup using multi-channel pipettes and allowing several thousand experiments to be performed in parallel by a single researcher. Second, the assay uses a population of worms in each well, so avoiding the well-to-well variation of RNAi phenotypes that is observed with single worm plate feeding protocols . We note that a related protocol has also been developed in the Plasterk lab .
We primarily developed this assay to facilitate the use of RNAi screens to systematically identify synthetic genetic interactions between genes. In this approach the RNAi phenotype of a gene in a worm strain carrying a defined genetic mutation (1) is directly compared to that in wild-type worms (2) and also to phenotype of the mutant worm strain alone (3) – a synthetic phenotype is defined here as a phenotype which is stronger following the double perturbation (1) than the expected additive phenotype of the two single perturbations (2×3).
As an alternative approach to identifying genetic interactions, two genes can be targeted simultaneously by RNAi (“combinatorial RNAi”) . The protocol we describe here works well for combinatorial RNAi, although it is much more effective when using an RNAi hypersensitive worm strain such as rrf-3 , eri-1 , or lin-35 [9,10]. Combinatorial RNAi is useful as it allows genetic interactions to be identified for genes for which a viable loss-of-function genetic allele is not available.
Finally, we note that the protocol as described here is not suitable for scoring phenotypes that can only be detected in adult progeny (for example behavioural phenotypes), because in a 50 µl volume progeny worms starve before reaching adulthood. However it is possible to grow progeny to adults by spotting each well onto seeded agar plates, or by increasing the volume of each assay, although this introduces more well-to-well variation.