Login

Join for Free!
119213 members


mRNA vs. cell reporter assay

Discussion of all aspects of cellular structure, physiology and communication.

Moderator: BioTeam

mRNA vs. cell reporter assay

Postby 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
aguy
Garter
Garter
 
Posts: 5
Joined: Wed Aug 03, 2011 7:51 am

Postby 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?
kk
Death Adder
Death Adder
 
Posts: 63
Joined: Fri Sep 23, 2005 7:58 pm

Postby 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.
User avatar
jonmoulton
Viper
Viper
 
Posts: 434
Joined: Fri Feb 15, 2008 5:38 pm
Location: Philomath, Oregon, USA



Return to Cell Biology

Who is online

Users browsing this forum: No registered users and 1 guest