Hi all,
I have a following problem. I have cloned a promoter of interest at 5´of Luc2 sequence in pGL4.10 vector (no minimal promoter, no enhancer). I transfect HEK293 cells with 0.5 or 1 ug of the plasmid DNA and 24hrs later I lyse the cells and measure luminiscence from expressed luciferase. Everything is ok, the bckgrd luminescence levels are low and the positive control is high enough. At 0.5ug of transfected DNA my promoter gives me a very nice signal: around 50 to 100 times higher then the Ctrl which is pGL4.10 vector alone without any promoter but at 1ug of transfected DNA the ratio is much lower (10-20 times) instead of higher. Why is that? Transfection with 1ug of DNA does not increase cell death (checked). I would appreciate any input in this case. Thanks!
My first guess would have been cell viability due to toxic levels of plasmid in the 1 ug samples. It could be related to your transfection method being less efficient with higher amounts of DNA (i.e. 1 ug of plasmid could be too viscous to efficiently be packaged into liposomes for lipofectamine transfections compared to 0.5 ug ---- the same could be said for too much plasmid lowering the efficiency for electroporations). More DNA is not always better.
Hi Daniel,
thanks for your input. In my case cell viability is not an issue. I have checked it several times. As for the DNA quantity I absolutely agree that more DNA is not always better, however I was able to actually transfect 2.5*105 HEK293 cells (Ca2+/Phosphate method, no Lipofectamine) with up to 5ug of DNA per well (6-well plates) in this setup and all seemed fine. For better understanding I attach an image of one of my latest results. Two Promoters were checked. Promoter 2 behaves as predicted while Promoter 1 shows a reduction of Luc activity when transfected with more DNA. The results shown here are means from three independent samples (triplicates). It doesn't make sense to me. What am I missing here?
It may be possible that your promoters behave differently in a stress-responsive sense. Perhaps one of your promoters is sensitive to silencing to avoid inclusion body formation and cell death under high transcription conditions, while your other promoter is insensitive. Is there a reason that you need to go so high on your DNA? Why not just use less and normalize to something like a co-transfected GFP?
Thanks Jordan,
I am setting up this experiment and it seemed reasonable quantity of DNA to use. Actually, I am scaling down already the DNA amount used in the assay due to this problem. I will tell you how it goes once I obtain new set of results. Jordan, do you think 100ng of DNA per well in a 6-well plate would be small quantity enough? Also, the two promoters I am studying are actually the same one but Promoter 2 has a SNP introduced compared to the Promoter 1 which is wild type in my experiment. Do you think that such a small change as 1nt within about 2kbps promoter could have such great effect on transcription?
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