We have used feeding RNAi to perform experiments on vulval development for years now, but recently found a disturbing shift. Starting in 2011, all RNAis fed to strain MT2124 let-60(n1046gf) substantially enhanced degree of induction. While we previously observed a slight but not significant induction with GFP-directed RNAi, now our induction is quite significant using the same GFP RNAi strain (streaked from the same -80 vial as before, so truly the same bugs). n1046 alone normally has a VPC score of ~4.3, and GFP-directed RNAi used to bump that up to ~4.6. Now we routinely see scores higher than 5, sometimes as high as 5.3. Same phenomenon but more potent?
We have used presumably neutral RNAis and luciferase-directed RNAi, and observed the same result. We have re-thawed MT2124 as well as using outcrossed n1046, and re-ordered MT2124 from the CGC. We re-made our feeding RNAi plates with all-new salts, cholesterol, Carbenicillin and IPTG, and even borrowed feeding RNAi plates from another lab: we always observed too much induction. We also re-transformed the GFP RNAi plasmid (L4440 background) into HT115.
Other sensitized hyper-induced worm backgrounds, like let-23(sa62)/+, are not hyper-induced by GFP(RNAi), so this phenomenon may be restricted to n1046. Alternatively, n1046 has always been the most sensitized background, so maybe n1046 is the canary in the coalmine for this phenomenon. Regardless, all RNAis cause the same hyper-induction phenotype in the n1046 background. Does anybody else have a precedent for this, in vulval induction or another process? And will anybody send me RNAi plates from another part of the country?
First thing we’ll try (hat tip Eric Lambie): ionic levels in media may have changed, particularly Mg. We’ll try varying MG levels in small batches. Second will be to make feeding RNAi plates using Noble agar.
You are saying that you measure the induction between fed worms and non-fed worms side by side? Did you try blasting the GFP RNAi to see if it may overlap with anything in the genome? Use lower stringency BLAST. The very factor that I see with n1046 induction is the temperature - even when sometimes I leave them outside.
Have you tried just feeding HT115 (no vector)? There is evidence that worms’ metabolism is strongly affected by the choice of bacterial strains, and you could be seeing synthetic effects in your mutant. See
My biggest concern is that feeding RNAi properties have changed. Previously, our gfp fRNAi or most other things would slightly hyper-induce, or not at all. Never statistically significant. Now we’re getting really strong hyper-induction. So some aspect of the system has changed, yet the clones, bugs, plate recipe and protocols have not changed.
Oh, and we’ve also done empty vector (L4440 alone, which should produce some nonsense dsRNA from the T7 vectors), and a new luciferase insert as a better control. Plus, L4440 inserts that have previously been neutral are now hyper-inducing.
Update: using +/- 50% MgS04 made no difference.
Abby: we have not tried HT115 alone without a plasmid, since we’d have to do so on non-RNAi plates. HT115 would not grow on standard IPTG+Carb plates. Also, I don’t think that variable has changed; same bugs as before.
And the use of Noble agar failed to fix our problem. It appears to not be impurities in agar or Mg concentrations. We’ll do some additional experiments with the Noble agar feeding RNAi plates, since their hyper-induction was less extreme than usual.
It appears to not be impurities in agar or Mg concentrations. We’ll do some additional experiments with the Noble agar feeding RNAi plates, since their hyper-induction was less extreme than usual.