The latest time point you can still expect to see an effect will depend on the growth rate of the cells, as the miRNA mimics get distributed over the daughter cells, and also on the half life of your protein of interest.

Using Qiagen's Alexa Fluor-labeled Allstars controls, mimics were, depending on the cell line, detectable for 72-96 hours after transfection, to disappear abruptly 24h later. The amount per cell would then have become too small to destinguish the fluorescence from the background.

I think 72 h and 96 h are quite sensible. I'm also doing the same thing using miRNA inhibitors and the then treating with chemotherapeutics.

Thanks everyone for your answers.Do you think I should treat my cells with small doses of chemotherapy 24 hours after the transfection or wait 48-72 hours after the trasnfection and then treat them with higher doses of chemo for 24 hours. In the first option the chemo effect will be slower allowing the mimics to work. In the second option the mimics will have more time to affect the proteins levels but their concentration in the cells will be much lower since they distribute over the daughter cells.

Well, I think it all depends on your experimental design. I would optimize my chemotherapeutics dosage over the time time course I plan to investigate, starting from 24h to the longest duration, to have a kill- curve, which will give idea of the IC50 of the chemotherapeutics, then I will stick to that concentration for the over- expression or knockdown experiments. At least, 48 h post transfection, you should be just good.

Thanks