RNAi feeding assay with mix of RNAi clones

Hi All
Is it possible to knock down multiple genes at once using RNAi feeding assays? If so, what is the upper limit of how many genes can be knocked down simultaneously in any of your guys experience?


Yes, it is possible.

I have done feeding RNAi by mixing two O/N cultures together at the same OD and seeding the mixture onto RNAi plates. I’ve only ever mixed two vectors together.

Some researchers have also made feeding vectors with multiple targets cloned into the same vector.


While it can work, it is also published that feeding multiple clones can cause the effects normally seen from feeding the individual clones to be attenuated or even to disappear entirely (one reference; a more recent report seems more optimistic about feeding two clones - though not three). This can be particularly confounding if the RNAi treatments you are combining normally have opposite effects when given individually. Using an RNAi-hypersensitive strain might maximize your chance of having both feeding clones work well; I think I vaguely recall a report to this effect, but can’t find it at the moment.

Injection RNAi of multiple clones is considered reliable, but is obviously less convenient than feeding.

Regarding the multiple-RNAis-in-the-same-bacteria approach, in addition to the paper Snug cites you might also check out Gouda et al’s report in the recent Worm Breeder’s Gazette, and their recent paper that they cite in the WBG report.