RNAi Knockdown

Greetings everyone. We are conducting RNAi knockdown (by mass feeding) for pos-1 gene recently in order to obtain sterile worms, and proceed with survival assay later on.
The eggs laid by the treated worms are not supposed to hatch. However, sometimes we did observe the presence of some small larvae after few days with the survival assay, meaning some of the eggs hatched.
Just wondering, there are possibilities where some of the worms are not properly silenced, meaning they didn’t feed on the E. coli?
Any advice on how to increase the efficiency of RNAi knockdown?
Thank you :slight_smile:

There are some simple things to look at:

  • Is the pos-1 dsRNA feeding strain expressing dsRNA effectively? At some point you induce with IPTG.
  • Are you feeding animals on plates, or trying to do this in liquid culture?
  • Are you feeding from hatching (L1), or starting with older animals? A few young adults or late L4s will result in progeny embryos that escape pos-1 knockdown.
  • Are your feeding strain and/or your plates contaminated with a bacteria other than E. coli HT115(DE3)?
  • Is your feeding strain still tet resistant, carrying the DE3 lysogen that has IPTG-inducible T7 RNA polymerase?


A possible control for efficacy is pop-1 to induce embryonic lethality. We use it all the time, and it is useful because we get escapers as RNAi efficacy goes down: old plates, or something wrong with the plate making process (say, adding IPTG or carbenicillin at higher temperature). We found that pop-1 is more sensitive than par-6, so we likes it better!

So we can get partial knockdown we sometimes keep some old feeding RNAi plates around. As Rasheed Wallace once said, “pop-1 don’t lie,” I made that up.

We will look into the suggestions provided, thank you so much :slight_smile: