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what is the best control for overexpression(luciferase assay)?

overexpression reporter assay luciferase

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#1 swchoi



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Posted 29 November 2011 - 03:55 PM

Hello, I am trying to find the response element in the promoter region of my gene. I pulled out the putative promoter region(1kb), insert into pGL3 basic vector and run duel-luciferase assay(promega). Here is my experiment setting: empty vector+1kb promoter, empty vector+pGL3 basic, overexpresison of gene A+1kb, overexpression of gene A+pGL3 basic. Renilla luciferase are all added as internal control.

Here is my problem: 1) no insert control(pGL3 basic) has a same trend of change as what I have seen in my gene A overexpression, to lesser extend, i.e., I do see 40% reduction of reporter activity by gene A overexpression when compared to empty vector, in both 1kb and pGL3 basic. Only difference is 1kb-pGL3 expressed cells has 'significantly' higher raw reading. I am confused. First, how come my pGL3 basic(no promoter in it) has 'some response' by overexpression? Second, what is the best way to normalize? Subtract pGL3 basic value from 1kb promoter insert value?

I suspect maybe reporter reduction is due to 'burden of cellular machinary' by overexpression, but not sure if it is true. If so, should l use GFP instead as my control to stress(?) cells somehow? But how do I know GFP won't interfere luciferase reading? And truly GFP be considered as better control for overexpression? Any suggestion for good control for overexpression?

Thank you so much for your advice!


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