First: What is S-TuDs? Cells, reagent? I do not know this name and found no real good hit by goole this.

To your siRNA question: What transfection reagent you are using? I am using Lipofectamine RNAiMAX and I do not replace the media. The cells treated with scrambled control siRNA show only 5-10% reduced proliferation after 72 hours of incubation.

With RNAiMAX or any other liposomal reagent the cells should have taken up the siRNA after 4-6 hours so over night incubation is enough. If you want to see phenotypic effects you should add 0.5 pMol siRNA for each well in a 96 well format and scale the siRNA amount up according to increased cell numbers in bigger well formats eg. 5000 cells in one 96 well plate well get 0.5 pMol siRNA, 50,000 cells in a 24 well get 5 pMol siRNA. Alsways control that you also upscale the volume of transfection reagent, 1 µl RNAiMax is sufficient to deliver 10 pMol siRNA! Look in the manual what your reagent can deliver.

If your gene is essential for the cells or in a signalling pathway you will see cell death after 16 h if it is really dramatic, or in 24 hour steps. It always depends.

I would recommend to replace the media when you add your retinoic acid then you can directly mix your acid in the media and all wells get the same concentration and you spare one handling step.

I hope this helped you. Regards

Similarly to Volker Baumann I do not know the S-TuD treatment.

For siRNA treatment I normally use Lipofectamine 2000 - in my case it is working better than RNAiMAX. However, you need to keep in mind that the type of cell culture might strongly affect transfection efficiency and you might need to optimize the best reagent for your experiments and the concentration of it! However, I would recommend you to start from the suggested amount according to the manufacture's procedure. Moreover, in my opinion you should also optimize the working concentration of siRNA.

For Lipofectamine 2000 I normally change medium 4 hours after transfection - Lipofectamine is toxic for some cell culture, e.g. human fibroblasts. However, for instance HEKs are less sensitive and they might be kept overnight with the transfection mixture without changing medium.

I hope my answer will shed some light on your situation:)

From my experience regarding gene silencing, I used both 1) retroviral/lentiviral vectors to generate a stable gene knockdown; and 2) siRNA for a transient knockdown.

1) For stable knockdown, I have used pSirenRetroQ (puro), pSuperRetro (puro or neo), pLKO.1 (puro). Usually your gene of interest will start to reduce its expression after 24 hours of infection with the retroviral particles; and after 48 hours you can get a satisfactory knockdown (more than 80% reduction in almost all cells)… and it could last weeks. If you are using a puro vector, selection happens in two days. This method is so efficient that I observe very little cell death during selection (that means that infection efficiency is very high). Sometimes, depending on the gene you are knocking down, you can see a decrease or increase in cell proliferation, for example. Phenotypic changes are dependent on the gene function on the cell. I particularly like stable knockdown because I can evaluate the cells in a long-term experiment, which is why it might be a good idea also for your case, cell differentiation. Let me know if you need a more detailed protocol.

2) For transient knockdown using siRNA, I use Dharmafect and 100 nM siRNA for 4 hours. Usually your gene of interest will reduce its expression after 24 hours and will last max 96 hours, with the lowest expression between 48-72 hours after knockdown. However, you would have to optimize those conditions for you specific cell line and gene. Again, if you need your gene to be silenced longer than 4 days, better to try a stable knockdown like I said before. Nevertheless, if you are performing a short-term experiment and you are ok with silencing your gene for 4 days only, siRNA would work just fine.

PS: S-TuD stands for "synthetic tough decoy RNA" (I found it here: http://www.ncbi.nlm.nih.gov/pubmed/19223327/; & here: http://www.ncbi.nlm.nih.gov/pubmed/22259037). In summary, S-TuDs are used to suppress miRNA activity. I never used those so I cannot comment on that.

Best of luck!