Materials

• Pur-A-Lyzer™ Midi Dialysis Kit (Sigma Aldrich #PURD60050-1KT)

Preparation

Prepare gene-specific dsRNAs as described in the dsRNA Synthesis protocol. Include a non-specific dsRNA control and a negative (no dsRNA) control.

Prepare parasite culture media as described in the Parasite Culture protocol. Scale in accordance with your experimental needs and the parasites used — it is recommended to make at least 400 mL of culture media (CM).

24 hours prior to the RNAi experiment

1. Parasites will typically arrive in 50 mL conical tubes. Remove nearly all of the shipping media. Pour the remaining contents, including parasites, into a clean 10 cm Petri plate containing sterile, pre-warmed CM. Using clean worm hooks, sort 3 adult parasites per well of a 12-well culture plate containing 3 mL CM per well. Label each culture plate with the date, sex and species of parasite, and your initials. Place culture plates at 37°C + 5% CO₂ for 24 hours prior to use.
2. Assemble and prepare the Pur-A-Lyzer Midi dialysis tubes:
   • Fill the Pur-A-Lyzer tube with 0.8 mL of sterile MilliQ water and incubate for at least 5 minutes. Empty the tube and ensure there is no leaking. Absorption of water by the dry membrane will cause a decrease in water level.
   • Load 800 μL of sterile, pre-warmed CM into the tube. Close with the provided caps — do not apply force.
   • Place the loaded tube in the supplied floating rack, then place the rack in a clean, autoclaved beaker containing 50 mL of sterile, pre-warmed CM. Place up to 3 tubes per beaker, grouped by treatment condition. Cover the beakers with foil and place in the 37°C + 5% CO₂ incubator overnight.

Day of the RNAi experiment

1. Fill a 6 cm Petri plate with sterile, pre-warmed CM. Remove the beaker containing the Pur-A-Lyzer tubes and the culture plates of adult parasites from the incubator.
2. Rinse the adult parasites under sterile conditions: use worm hooks to gather one group of 3 adult parasites and transfer them to the small Petri dish containing CM. Allow to rinse for a few seconds.
3. Remove the foil cover from the dialysis beaker and remove the tube holder using clean, sterile forceps. Remove the cap from a Pur-A-Lyzer tube.
4. Add the desired volume of dsRNA to the tube, for a final dsRNA concentration anywhere from 50 μg/mL to 2 mg/mL. Consult the literature.
5. Gather the group of adults soaking in the Petri dish using the worm hooks and transfer them to the Pur-A-Lyzer tube. Ensure all parasites are submerged in the CM/dsRNA. Close the tube with the cap. Do not put more than 3 adults in each tube.
6. Repeat steps 2–5 until each desired group of 3 adult parasites has been added to a tube.
7. Discard the CM in the beaker and replace with 100 mL of sterile, pre-warmed CM. Place the loaded tubes, in the floating rack, back into the beaker. Cover with foil and place in the 37°C + 5% CO₂ incubator.
8. Allow the tubes and parasites to incubate for 72 hours. Check the beaker and media for signs of contamination or colour change.

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If contamination is noted, obtain a new autoclaved beaker, fill with 100 mL of sterile pre-warmed CM, and transfer the rack of tubes using sterile forceps. Cover with new foil and return to the incubator.

If the CM surrounding the tubes changes colour, that is a sign of pH change and the CM needs changing — discard the old CM and replace with sterile, pre-warmed CM.

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72 hours post-soaking (knockdown check)

1. Remove the beaker from the incubator and move to the BSC. Remove the holder containing the tubes, remove the cap, and remove the parasites using worm hooks. Wash the adults in a Petri dish containing sterile, pre-warmed RPMI amended with 1% (v/v) P/S.