Currently, I wanted to perform an experiment where I have to perform a drug treatment and siRNA transfection together. What shall be the way to design the protocol? Shall I perform the drug treatment first followed by the siRNA treatment thereafter or can I do both of them simultaneously? Actually, the drug causes nuclear localization of my target protein after 72 hours and there is another protein responsible for the cytoplasmic localization which I want to knockdown, down so as to see the majority within the nucleus. What shall be my actual approach?
For these types of questions, it is usually helpful to be clear about what scientific question you are trying to answer by doing this experiment.
If I understand your experiment correctly, you want to knock down "a protein responsible for the cytoplasmic localization of your target protein". Let's call this "protein C" and your target protein "protein T". The drug causes nuclear localization of protein T. What is the question you're trying to answer here?
As a very general principle, in a siRNA knockdown experiment you will want sufficient time for the mRNA knockdown to result in a decrease in the level of the protein (in this case, protein C). If you knock down C mRNA, do you know how many days it takes for protein C levels to fall?
Hi,
You could try transfecting your cells with siRNA and changing the medium after 6-8 hours. While changing the medium you could treat your cells with the drug. Though, it is important to know how long your siRNA maintains downregulating the gene. Firstly, I would recommend you to perform the transfections and check the downregulation at different time points (protein level detection is better). Hope this helps!
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