Hi, I have a GFP reporter cell line that drives GFP expression upon activation of a transcription factor. In theory, I can study transcription factor activation via the GFP signal, or by measuring the mRNA of the transcription factor. I wonder what are the pros and cons of each. Can anyone help me figure this out?
I have the same question, phrased in a slightly different way:
Maybe the reporter system provides us with more information than RT-PCR/Western blot, because it proves that the transcription factor is actually active too, not just present? I thought it is enough to detect a significant increase in the expression by Western blot and we could conclude that the particular transcription factor is more active… do TFs need activity assays to be validated?
If the cells autofluoresce, this could confound interpretation of the GTP signal. if you are looking for downregulation of transcription, it takes a while for preexisting GPR to degrade but the mRNA signal is likely to disappear fairly rapidly once transcription stops. For upregulation, mRNA will reach steady-state fairly rapidly but there will be a longer delay as enough GFP is translated to reach a stable level.