I have been using beta estradiol and checking for its effect on a particular gene promoter region. Treatment initially showed me a multifold increase when compared to promoter activity without estradiol treatment. Lately, in the experiment being repeated, I am not observing any change between treated and untreated. I make fresh working stocks of the solution of estradiol in DMSO/Ethanol (powder from sigma with a 2 year shelf life at room temp). Other than using a separate ERE-Luc plasmid to check for the working of estradiol, are there any other methods to check whether treatment is working or not?
First this will depend on the cell line or cell type you are using, may be one can find some well know target gene for betaE in your cells (check in the literature for your model). Second, you made fresh betaE solution but this discrepancy could also be due to the number of passage of your cells (if you use cell lines), the state of the cells (e.g. confluency, mycoplasma, etc...)
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