mRNA vs. cell reporter assay

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aguy
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mRNA vs. cell reporter assay

Post by aguy » Mon Jul 07, 2014 2:50 am

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

kk
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Post by kk » Mon Sep 08, 2014 2:57 pm

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?

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jonmoulton
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Post by jonmoulton » Mon Sep 08, 2014 5:27 pm

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.

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Post by Fornita » Mon Jun 13, 2016 9:58 am

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