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

overexpression reporter assay luciferase

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#1 swchoi

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!

Best,
Sung





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