- To begin, add carbenicillin and one millimolar IPTG to control and test culture flasks containing S basal media. Spin a tube containing larvae at growth stage one, also called L1 larvae, for a few minutes. Now remove the supernatant and place two microliters of L1s on an agar plate. Place the plate under a dissecting microscope and count the number of larvae.
Add RNAi bacteria carrying a non-specific RNAi plasmid to the control flask and bacteria with gene- targeting RNAi to the test flask. The amount of bacteria added should be proportional to the number of worms.
Let the larvae grow with continuous shaking to ensure proper oxygenation. The larvae feed on the bacteria. Inside the larva, the small interfering RNA binds to the complementary mRNA produced by the targeted gene and inhibits protein expression. Finally, examine the worms for your phenotype of interest.
In the example protocol, we will treat L1 larvae with RNAi targeting the daf-2 gene, in liquid culture.
- To begin treatment, add 207.45 milliliters of S basal media with additional reagents as described in the accompanying text protocol to a 2,800 milliliter Fernbach culture flask. Add a final concentration of 50 micrograms per milliliter of carbenicillin and 1 millimolar of IPTG, and close the flask with a membrane screw cap.
Take the L1s out of the 25 degree Celsius incubator and transfer them to 15 milliliter tubes. Centrifuge the L1s at 1,900 times g for three minutes. After spinning, remove the supernatant. Under a microscope, count the L1s per two microliters, and average the numbers obtained from at least nine drops.
Next, add 50,000 worms for the young worm collection and 100,000 worms for the aged worm collection into four Fernbach culture flasks prepared in the previous step. Then add control RNAi bacteria and RNAi bacteria for the gene of interest proportionally to the number of worms.
After adding bacteria, complete worm culture with S basal to bring the total volume to 300 milliliters. Incubate the worm culture at 25 degrees Celsius in a shaking incubator with 150 RPM until collection.