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    • #17846
      aguy
      Participant

      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?

      Thanks,
      John

    • #115422
      kk
      Participant

      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?

    • #115424
      jonmoulton
      Participant

      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.

    • #116021
      Fornita
      Participant

      These two assays could be complement for each other, you could not get a conclusion just from one assay.

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