Protocol for RNAi by feeding on plates

Model Organism: Worms Methods & Reagents
1

Hi everybody,

I have a question about RNAi by feeding on plates. I’ve had some troubles recently to make my experiments working and I found different protocols in the litterature. So I’d like to have some advice of people who have the experience of that type of experiments. What protocol works best for you? Do you use fresh plates/ plates that have few days? Do you induce the bacteria before seeding them? If so, for how long? And what RNAi do you use as a control?
Thanks a lot for your help.

KR

2

We generally use fresh plates <2 weeks, don’t induce before seeding and use die-1 as a control which should lead to embryonic lethality

3

i have stuck quite firmly to the protocol available in wormbook and from the Ahringer lab website. with some slight modifications, RNAi always work:

  1. RNAi plates with 1mM IPTG and 50ug/ml Ampicillin made fresh, but let them dry at room temp for at least 4 days before seeding RNAi clones and adding your worms. after 8 days of drying, keep them at 4 degrees and I can use them up to 1 month.

  2. Grow RNAi clones in LB+50ug/ml ampicillin 7-8 hours before seeding on RNAi plates and let dry.

  3. RNAi clones are recovered fresh from stocks once every month on LB + 50ug/ml amp + 15ug/ml tet. keep them in the fridge.

  4. depending on the experiment, but i often bleach my worms and add the synced L1 or unsynced eggs straight onto seeded RNAi plates.

  5. i use unc-22 as a control for N2 worms.

hope this helps :slight_smile:

4

Thanks a lot for your replies! :slight_smile: I’ll try this and see if it works ok for me.
Do you usually observe RNAi effects in all the worms/ most worms? In the paper from the Ahringer lab (Kamath et al, 2000), they observe 100% phenotypes. Do you have the same efficiency? I guess that probably depends on the gene and the phenotype.

5

We always induce with IPTG for 4 hours before seeding, as this seems to forgo the requirement to have fresh plates that are less than 2 weeks old. There are obviously many ways to do these type of experiments, important thing is to find one that works and stick to it! Good luck

6

Thanks for your reply medawg!
I think you’re right but the hard part is to find the way that works :wink:

7

yes depending on the gene and length of RNAi treatment as well as all sorts of factors ranging from how well you prepare the worms and your plates and your RNAi bacteria clones etc. also, knocking down the gene at particular development stages has different efficiency and effects too (knowing the requirement for your gene to manifest a phenotype during pre/post-embryonic stages per se).

unc-22 is pretty nearly 100%, approximately 48h of unc-22 RNAi treatment from bleached eggs/L1 worms.

genes in the neurons are known to be hard to knock down.

hope your RNAi works! rem to always include a phenotypically-obvious control :slight_smile:

8

Thanks for your comments. I’ll give it a try and let you know how it goes :wink: