What is the most sensitive assay for promoter strength? - dual luciferase assay? GFP? SEAP assay? Miller or something else? (Aug/11/2008 )

Hi,
I'm looking for a very sensitive assay system for measuring the strength of different promoters.

So far I've looked at using GFP + fluorometer, dual luciferase assay (Promega) + luminometer, and SEAP assay (Clontech) also chemiluminometer or Beta-Galactosidase/Miller assay. I want to know which of these is the most sensitive as I'm interested in measuring a number of weak promoters. My initial reading tells me that GFP would not be sensitive due to background fluorescence. I cannot find any comparison of the sensitivity of the SEAP assay with the dual luciferase assay, but it seems that the dual luciferase assay would be better because it has an internal control whereas the SEAP or Miller do not appear to. Do you agree with my selection of dual luciferase assay as the most sensitive? Are there other assays our there that might be more sensitive, or others that also provide an internal control?

cheers,
Philis

luciferase assay is a time-tested procedure for comparing promoter strengths........Mind you, I have never done it myself! But I agree GFp just produces tooo much background.

Hi Philis,

If you are looking for a sensitive reporter for a weak promoter then your best choice would be a secreted reporter like SEAP. Have you thought of the secreted luciferase from Clontech? I think they call it metluc.
I have been very successful with that (borrowed from another lab ofcourse:)) I guess because the reporter is secreted and not subjected to degradation outside the cell it allows accumulation over time. I was happy with it. They do say that you can multiplex it with SEAP. Ihave tried renilla but was not happy with accumulation over time. Have not tried B-gal but I don't see how that would be different.

Antonio

Hi Antonio,

Thanks, I looked into the Clontech MetLuc as you suggested, (http://www.clontech.com/images/ctq/OCT07UPD/CR7X2506_RTG_FAQ_US.pdf) however I can't see a way to use an internal reference there?

Can you explain more about what was wrong you were not happy about with the renilla please? I'll have a think if it is more important to have an absolute measurement or a relative one as I think this will help me decide which is best.

Thanks Philis