RNAi inconsistencies

Model Organism: Worms Methods & Reagents
1

I am having problems with consistent KD using RNAi…some trials I get great KD, others I get none. The general protocol I’m following is:

  1. Seed LB+carb plates with fresh-grown stock; let grow 2-3 days
  2. Add 0.1MIPTG/50ug/ml carb to seed approx 1 hr before picking worms to plates
  3. Pick L4 worms to plates, allow to lay eggs, remove the following day
  4. Allow eggs to mature, gather worms for downstream purposes (RNA, Westerns, etc)

Any advice would be greatly appreciated, as I’m running out of things to trouble-shoot!

2

Hi,

From what I can understand from your post, you add carb and IPTG to the bacterial culture to induce RNAi. We usually add carb and IPTG to our media itself after autoclaving (allowing media to cool sufficiently). Next, we seed our RNAi culture and dry the plates. We also wait at least 10-12hrs before seeding our worms to allow sufficient time for IPTG induced expression of dsRNA in bacteria. This might help address inconsistencies in your knockdown.
Hope this helps

3

You can also induce in liquid culture for a few hours before plating onto +IPTG/Carb NGM plates.

4

if you want a consistently robust response, don’t have lots of different clones or need millions of animals, then injections are a good option.

5

I can confirm that RNAi by injection is really reproducible. If you have few gene to test it’s a good alternative.
A post-doc in our lab did a targeted screen (around 100 candidates) by injection and got result in several weeks.