RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 103

12. Extra L1 larvae from day 3 can be used to seed extra-large plates for the
following week ' s experiment. In our lab, we plate 10,000 L1 larvae on each
extra-large plate, and we prepare four plates for each strain. This produces
enough L1 larvae to dispense more than 200 flat-bottom plates and to seed
NGM plates for the following week ' s experiment.
13. To count the number of hatched L1 larvae in the suspension, dilute 100 m L
of larvae suspension with 900 m L of M9, and mix well. From this dilution,
place three drops of 10 m L on the lid of a medium petri plate. Count the
number of larvae in each drop, and calculate the dilutions necessary to
achieve a final concentration of 10 larvae/20 m L. Dilute with complete
S-Basal. One flat-bottom plate requires about 2 mL of larvae suspension
to fill all wells.
When calculating the volume of larvae suspension to dilute, consider that the
Wellmate 1 has a 10 mL dead volume in the cartridge, and depending on the
diameter of the container used to dispense the worms, extra volume is needed to
avoid aspirating air into the tubing.
14. To dispense the larvae suspension, we use a small sterile beaker or a disposable
sterile plastic container with a stirring bar to continuously mix the suspension.
This keeps the suspension homogeneous throughout the process. We have found
that with the dilution we use, 92% of our assay wells show a range of 6-16
worms, and all look healthy.
15. Assemble the humid chambers using plastic Tupperware containers lined with
wet paper towels. It is important to make sure that the containers are hermet-
ically sealed with Parafilm, especially if incubating at higher temperatures, to
avoid any potential dehydration of the samples.
B. Scoring of Interactions/Phenotypes
1. Visual
Before scoring the images, define the variables to be evaluated and the method of
evaluation. As a general guide, we describe below the criteria we have developed in
our laboratory to score genetic enhancers and suppressors, though each laboratory
will most likely want to develop customized assay protocols for their own purposes.
For each experiment, we analyze two technical replicates (test plates A and B)
with the ts mutant fed on RNAi bacteria targeting a gene of interest (''double-knock-
downs''). These are compared to three controls: N2 strain fed on the empty vector
bacteria L4440 (WT worms), N2 fed on the same RNAi bacteria as the test plates
(effect of the RNAi alone), and the mutant strain fed on L4440 (effect of the
mutation). To facilitate scoring, we usually open a set of images from the same
experiment together (this can be done using a variety of common image visualization
programs), which allows us to scroll quickly through the results of all 96 wells from
the same plate. We record all phenotype scores in an Excel worksheet designed to
mimic the layout of a 96-well plate.