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Dual luciferase assay - proper controls? - LUC below pGL3 despite protein activity (Jun/22/2011 )

Hey everyone,

I am trying to perform a dual luciferase reporter assay in a cell line with promoters established from another cell line.
cDNA and protein of the gene are present in both cell lines.

Problem: In the new cell line, the luciferase activity for my promoters of interest is below pGL3, whereas TKpGL3 is as highly active as in the other cell line. Renilla activity was equal in all probes.

So I am looking for appropriate controls, to find out if this is sequence specific. Thought about beta-actin or GAPDH or any other gene naturally expressed in the cells...?

If someone has any ideas or suggestions on how to find out what's going on I'd be very grateful!

Thanks!!!

-greenhorn-

If I have understood correctly:



<*>equal Renilla tells you that the transfection efficiency is similar.
<*>high Tk-luciferase tells you that the system is working.
<*>your sequence gives lower values than the empty vector.


Are you sure that you are putting equimolar amounts of your promoter-luc and empty-luc? If you just put the same nanograms, and the promoter turns out to be not active, being the empty vector shorter, you are putting more empty vector, so you could expect more RLU.

If the results are tru, it looks like something is silencing your sequence. To check if this is sequence-specific you may want to try with a scrambled sequence.

You can read more luciferase misfacts here.

-96well-

96well on Tue Jun 28 13:57:53 2011 said:


If I have understood correctly:



<*>equal Renilla tells you that the transfection efficiency is similar.
<*>high Tk-luciferase tells you that the system is working.
<*>your sequence gives lower values than the empty vector.


Are you sure that you are putting equimolar amounts of your promoter-luc and empty-luc? If you just put the same nanograms, and the promoter turns out to be not active, being the empty vector shorter, you are putting more empty vector, so you could expect more RLU.

If the results are tru, it looks like something is silencing your sequence. To check if this is sequence-specific you may want to try with a scrambled sequence.

You can read more luciferase misfacts here.


Thank you a lot for your reply!
The Renilla is for normalization of the expression value and tells me that the transfection itself worked in a comparable effciency, as I understood. Yes, the TK-luciferase tells me that the luciferase itself works and can be detected. And yes, my sequence gives lower values than the empty vector, even if it is not in pGL3 but in TKpGL3. And the amounts are equimolar, they also worked in another cell line, where the essay was established. I thought maybe there is some kind of alternative promoter in this cell line for the same gene (farer away)? The RNA and protein are present and active.
How can I get a scrambled sequence? Thanks again so much for your help!

-greenhorn-

you can scramble your dna here (shuffle DNA) and then go for gene synthesis

-96well-

Thanks a lot!! Hope can make it work now...

-greenhorn-