optimum time for assay of target gene - siRNA (Sep/17/2008 )
i am doing transfection on MDCK cells, using SIPORT-Lipid form Ambion, please suggest me how much time should be given for transfection and after how much time the assay for target gene can be done.. further why it is advised not to use antibiotics during transfection and what is it's mechanism.
Here are some factors to consider when determining the timing of the assay for knockdown. Partially, this depends on the nature of the assay you will be using. The concentration of the target mRNA may change rapidly, but the concentration of the preexisting protein will slowly decrease through normal protein turnover but without replacement by new translation (or with reduced translation, depending on the knockdown efficacy). This means that an RNA-level assay can be done fairly quickly, likely limited mainly by the rate of delivery of the knockdown reagent. However, immunoassays for the target protein must be delayed to allow degradation of enough preexisting protein that the decrease in protein signal can be detected. If your interest is in determining when the cells are undergoing physiological consequences of the knockdown, you need to wait at least as long as it takes for the protein signals to be suppressed.
I can't help with your other transfection questions, as I don't use that reagent.