n=3 for pSILAC - (Sep/14/2017 )

Hi,

I would like to compare de novo protein synthesis in my wt and KO cell line using pSILAC. Our mass spec people want 3 biological replicates. Usually when I do cell culture experimets, I plate cells one day, do the whole experiment, then repeat twice more. Then I call this n=3 biological replicates. But our mass spec guys say I can just plate everything in triplicates, do everything one day, and as long as the triplicates don't mix, I can call these biological replicates and have n=3. In my book, these are technical replicates. Their logic is that any "omics" experiments are so expensive and time consuming to repeat 3 times individually, so no reviewer will ask me for a repeat experiment, as long as I have 3 "biological" replicates it is ok. They also say that even if I were to set it up the way I usually do, and just run all the samples in the mass spec at the same time, the biological noise I have in my set up will eat up any significant, real difference I have.

I am not sure what to do...

Realistically how independent are your replicates anyway? - are you using the same stock of cells to set up each replicate (i.e. do you get up fresh cells each time you do a replicate or are you using the same ones at a different passage)?. Do you use the same bottle of medium? same bulk reagents for each (PBS, transfection reagents etc.)?

You can make them biological replicates by setting them up independently - i.e. make up the reagents for each replicate separately - for instance if you were doing a transfection you would prepare the DNA/transfection reagent mix separately for each replicate. You could also have separate flasks of cells to prepare the seeded cells from, so as to make each replicate more independent