Has anyone here done a starvation assay ? I found these two papers online that talk about it in materials and methods section. I am, however a bit confused since I am not clear how long you keep the worms in plates without food and peptone (for certain hours or tlil they die?). Any input is appreciated
thanks in advance
in one of the papers you linked to;
under the heading;
‘IFG-1 Is Downregulated under Nutrient-Limitation Conditions and Modulates Survival under Starvation’
the authors say that they starved the worms for 48 hours (and also tested 0, 24 & 72 hours, see the supplementary figure).
The lifespan assays (until they die) were undertaken on both starved and non-starved worms to assay the expression (and therefore role) of particular stress response genes in the mutant they were interested in.
Hope that’s clear now.
Steve nailed it on the head.
Here are some tips for actually doing the assay.
We use large (100mm) plates without peptone and plate 30-40 worms per plate. Make sure there are absolutely no bubbles in the agar and I would recommend pouring the plates with an automatic pipetter to ensure consistent volume. I usually put 25mL per plate. This is strictly because finding the focal plane is sometimes a pain and if all of your plates are a consistent volume it makes finding the worms easier.
Know exactly how many worms are on each plate so that when you’re scoring you can find them all.
Worms get transferred once a week to fresh plates. You don’t want to risk them drying out and forming small cracks that the worms can burrow in to. Transferring is a pain because you can’t use e.coli on your worm pick (otherwise it defeats the purpose of the starvation assay).
P.S. Starvation lifespans can last quite a while so be prepared for a lengthy assay. Keep an eye on them frequently because there’s nothing worse than being a month in and having to censor entire plates because they all burrowed.
Thanks Steve and Matt Your response greatly clarifies my doubts. Just to be sure with what you said, I am testing RNAi for my gene of interest to see whether it affects starvation. Matt, If I am understanding what you said correctly, I starve my RNAi fed worms by moving them to 100mm RNAi plates without peptone but WITH RNAi of my interest and then compare it to control RNAi
You don’t want any bacteria on the starvation plates. So I’m guessing your assay would go roughly something like:
(1)Place worms on RNAi bacteria of choice for some duration to ensure knockdown of your gene of interest.
(2)After that duration of time, move worms to starvation plates where there is absolutely no bacteria at all and starve your worms for however long you’d like to test (this can include indefinitely).
(3)(Optional) Move worms back to RNAi plates.
I would recommend growing up a large number of worms on RNAi (in excess of what you need), washing them 2-3 times in S.Basal or M9 and then transferring from buffer to the starvation plates. The wash ensures that no food is transferred along with the worms to the starvation plates and you’re doing it in excess to account for worms that adhere to the tubes or pipette tips during the wash and transfer.
You’ll need to fine tune the procedure in order to elucidate whatever it is you want to learn about obviously.