Hi,
I am performing RNAi by feeding in several genes on N2 strain (from L1 and at 25ºC),
as a negative control I am using HT115+L4440 (Empty vector).
I let animals growing on RNAi plates from L1 to L4 and then I transfer individuals onto single RNAi plates to score for phenotypes (Brood size)…
Is the third time I try this protocol (using different glycerol stocks for Empty vector) and I am always getting an average around 130 larvae for the negative control… As I am observing reduced brood size for the genes I am studying, I cannot obtain definitive results as I still getting low progeny levels with the negative control.
I am always using new batches of N2 animals in every experiment…
Has anybody observed the same when using Empty vector as a negative control? Could be an effect of the temperature (25ºC)? Should I maybe use GFP RNAi?
You might also consider whether brood side on HT115 is different then on OP50. I’m almost sure this has been looked at but can’t remember off the top of my head whether there is a significant difference. Good luck.
The thing is that for the control ( HT115 ) I’m observing a Rbs (around 150 worms) while upon RNAi’s against my genes of interest I can observe a reproducible brood size higher than the control (170, 180 worms) …
I was suggesting that you include an HT115 control that had no plasmid whatsoever to compare to your empty vector control. If you see a difference, then your empty vector isn’t actually empty. If you actually do the comparison, you might as well throw in an OP50 control too.
Temperature was previously mentioned, it’s a good point that’s worth thinking about too.
I’ve heard of the same thing in my friend’s lab.
Brood size is decreased in empty vector HT115+L4440, but was normal in worms fed GFP RNAi though, so they used that instead.
