I have been doing RNAi experiments to knockdown gene A’s expression. I have noticed that the RNAi experiment sometimes works but sometiimes it just doesn’t work. I used exactly the same protocol and the same age worms. I am really frustrated by the totally different results :(. My control gene’s RNAi works everytime. Has anyone met the similar problem before? could anyone tell me why I saw these kind of results? any possible reasons? Thx!
Hi Kim!
We have been having the exact same problem, but we can’t find out why. We have changed the antibiotic and IPTG concentrations, with no different results: controls with dpy-11 and unc-22 work alright but our experiment sometimes does and sometimes doesn’t. We don’t either know why.
Thanks for sharing your experience with me. Now at least I know I am not alone on RNAi experiments in terms of success or not. Is that possible this is caused by gene redundancy?
I am not sure how you are doing your experiment exactly, but I have found that adding IPTG to the cultures for 2 hours before seeding the feeding plates helps your consistency from experiment to experiment.
I usually make some NGM plates (about 100) with AMP and IPTG. Stock them at 4 degree for a period of time.
In your case, my understaning is that you add IPTG to your bacterial culture. Could you let me know the final concentration of IPTG after you add it to bacterial culture?
I have never done RNAi experiment this way. I will try this method next time. Thank you!
Do you also add IPTG to your feeding plates?
Here is what I normally do. I do have IPTG in the plates as well, but I think that is where the variablity comes into play, the IPTG concentration in each plate from each pour I think works slightly differently.
(Day1)
Pick a colony up and independently culture in 2xYTB (3 ml) w/ Amp & Tet for O/N (12-14 hrs). I usually pick colonies from plates that are less than 3 weeks old.
↓
(Day2)
transfer 30 μl of culture to New 3 ml 2xYTB w/ Amp & Tet.
↓ culture for 2.5 hrs@ 37℃
↓ add 10 ul of 100 mM IPTG
↓ Induction for 4 hrs
(I usually let the NGM plate containing IPTG&Amp&Tet get back to room temp during the induction. Keep the plates in dark.)
↓seed (2 drops and spread them! ) onto the NGM plate containing IPTG&Amp&Tet. Place plates O/N at 37 deg.
hope this helps!
Hi medawg,
Thank you for sharing your protocol. I have never used 2X YTB medium before, so could you let me know the receipe of how to make such medium? (I usually use LB)
Thanks!
Kim
for 1L of 2xYT:
16g bacto-tryptone
10g yeast extract
5g NaCl
Adjust to pH 7.0 with 5N NaOH
I normally grow OP-50 in 2xYTB instead of LB. as you can see from the receipe above it has more nutrients than LB.
Hello. Nattha, please clarify about the Kanamycin resistance of HT115. Do you have an exact genotype for HT115 to share? The CGC has “Bacterial strain. Genotype: F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse III minus). This strain grows on LB or 2XYT plates. This strain is tetracycline resistant. Researchers using this strain should test for expression by transforming in one of the plasmids from the Fire Vector Kit (1999) (pLT76, e.g.) using standard CaCl2 transformation techniques.”
There doesn’t seem to be an explicit reference to Kanamycin.
I think they meant Tet resistance, not Kan :o
Oops sorry I was mistaken. It’s Tet marked not Kanamycin. Anyway OP50 still expresses RNaseIII so RNAi with it just won’t work.
When I do solid RNAi, I induce it in NGM agar at room temperature.
I was referring to the use of 2x YTB in both RNAi experiments and OP-50 bacteria growth for regular worm experiments.
normally i prepare my RNAi bacteria on fresh NGM/IPTG plate… from what my senior told me, they said that the NGM/IPTG plate wont last more than 2 weeks, might be due to the short half-life of IPTG?
I suspect that might be part of the reason why it works sometimes and not others, but if you induce in culture it is more consistent, cause you are always using fresh IPTG.
It’s not good to store RNAi plate at 4 degree for uncertain time. Prepare fresh RNAi plate as possible as you can, or add IPTG some hours before you seed worms.
We’ve stored RNAi plates at RT for up to a week and in the cold room up to a month and never had an issue. The only consideration is to store the plates in sealed bags so that the plates don’t dry out.
One alternative for your consideration is the age of the bacterial colonies. We’ve found that some RNAi clones are not well maintained. In the most severe cases a clone can go bad after two weeks, but that is the exception. Our rule of thumb is that the clones are good for a month (stored at 4C) and after two months I wouldn’t trust them at all. With that said, I highly encourage the use of bacterial clones as early as possible.
We always use pop-1(fRNAi) as a positive RNAi control, and if we don’t get 100% lethality we throw the whole experiment out. pop-1 is a good benchmark for efficient feeding RNAi.
We have never had problems with stored fRNAi plates less than a month old. I’ve even gotten 100% pop-1 lethality from plates 4 months old, but to be cautious we never actually use plates older than a month.
We also never use fRNAi cultures older than 2 weeks, usually 1 week. Finally, for RNAi we ONLY compare data gathered in the same experiment, since variability has always been an issue to some degree. Very conservative and cautious is your best bet.