Dialysis Tube RNAi

Materials

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

Protocol

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

Prepare parasite culture media as described in the Parasite Culture protocol. Note: You will need to scale in accordance with your experimental needs and parasites used. It is recommended to make at least 400 mL of culture media (CM).

24-hours prior to RNAi experiment

  1. Parasites will typically arrive in 50 mL conical tubes. Remove nearly all of the shipping media. Pour the remaining contents of tube 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 37ºC + 5% CO2 for 24 hours prior to use.

  2. Assemble and prepare the Sigma Aldrich Pur-A-Lyzer Midi Dialysis tubes (PURD60050-1KT)

    • Fill the Pur-A-Lyzer tube with 0.8 mL of sterile milliQ water. Incubate for at least 5 minutes. Empty the tube. Ensure there is no water leaking from the tube. Absorption of water by the dry membrane will cause a decrease in water level.

    • Load 800 µL of sterile, pre-warmed CM into the Pur-A-Lyzer tube. Close the tube with the provided caps (do not apply force).

    • Place the loaded Pur-A-Lyzer tube in the supplied floating rack and then place the rack in a clean, autoclaved beaker containing 50 mL of sterile, pre-warmed CM. Place up to 3 tubes in a beaker, grouped by treatment condition. Place a foil cover over the tops of the beakers, and place in the 37ºC + 5% CO2 incubator overnight prior to use in RNAi experiment.

Day of RNAi Experiment

  1. Fill a 6 cm Petri plate by placing sterile, pre-warmed CM. Remove beaker containing Pur-A-Lyzer and culture plates containing groups of adult parasites from the incubator.

  2. Rinse the adult parasites under sterile conditions, by using worm hooks to gather one group of 3 adult parasites and transfer to the small Petri dish containing CM. Allow to rinse for a few seconds.

  3. Remove foil cover from dialysis beaker, remove tube holder from the beaker using clean, sterile forceps. Remove the cap to a Pur-A-Lyzer tube.

  4. Add the desired volume of dsRNA to the tube for a final dsRNA concentration of anywhere from 50 µg/mL - 2 mg/mL (consult literature).

  5. Gather the group of adults that have been soaking in the Petri dish using the worm hooks. Transfer the parasites to the Pur-A-Lyzer tube. Ensure all parasites are submerged in the CM/dsRNA within the tube. Close the tube with the cap. Do not put more than 3 adults in each Pur-A-Lyzer tube.

  6. Repeat steps 2-5 until each desired group of 3 adult parasites have been added to a Pur-A-Lyzer tube.

  7. Discard the CM in the beaker. Replace with 100 mL of sterile, pre-warmed CM. Place the loaded Pur-A-Lyzer tubes containing parasites in the supplied floating rack back into the beaker. Place a foil cover over the top of the beaker, and place in the 37ºC + 5% CO2 incubator.

  8. Allow the tubes and parasites to incubate for 72 hours. Check the beaker and media for signs of contamination or color change.

    Note: If contamination is noted, obtain a new autoclaved beaker. Fill with 100 mL of sterile, pre-warmed CM. Transfer the rack containing the Pur-A-Lyzer tubes to this new beaker using sterile forceps. Place a new foil cover over the top of the beaker, and place back in the 37ºC + 5% CO2 incubator. If the CM surrounding the tubes changes color, this is a sign of pH change, and the CM will need to be changed. Discard old CM and replace with sterile, pre-warmed CM.

72-hours Post-Soaking (Knock-down check)

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

  2. After washing, using worm hooks, transfer the adults to a sterile, labelled 1.5 mL Eppendorf tube. The adults should stick to the inside of the tube. Using a p1000 pipettor, quickly pipette 100 µL of sterile RPMI on the group of adults on the inside wall of the tube. This should draw all the adults to the bottom of the tube. A brief spin in a benchtop centrifuge can also help if adults have not been drawn to the bottom.

  3. Repeat the above steps until all desired groups of adult parasites have been washed and transferred to a sterile, labelled 1.5 mL Eppendorf tube.

  4. Snap freeze the tubes in liquid nitrogen. Store at -80ºC.

    Note: If time allows, perform RNA extraction immediately.

  5. Measure gene knock-down using qPCR protocol.

RNAi Assay Optimization

  • If significant transcript knock-down (> 50%) is not achieved within 72 hours, you can consider increasing dsRNA concentration, repeated exposure to dsRNAs (replenishing every other day), or increasing dsRNA exposure through extending the timeline of culture.

  • If significant transcript knock-down (> 50%) is achieved, you will need to align the dynamics and timing of this knock-down to measuring your phenotypic endpoint(s) of interest.