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Helping for dual-luciferase assay


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#1 neuronzy

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Posted 14 March 2010 - 09:13 PM

Hello together,

I perform dual-luciferase(Promega kit) for comparison between two different transfected transcription factor p53, one wild-type and the other is H179R mut p53, which is supposed to have 10-fold less activity compared with the wild-type. My result showed ~7 fold decrease in the transactivity of p53 H179R, but the firefly luciferase values are similar between the two p53s, while it's the Renilla luciferase value of H179R p53 transfected wells increased ~7 fold. Does anyone have similar experience or some suggestions? Every suggestion would be helpful for me, and thanks very much!

Regards
Kevin

#2 Dr Teeth

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Posted 15 March 2010 - 09:55 AM

Hello together,

I perform dual-luciferase(Promega kit) for comparison between two different transfected transcription factor p53, one wild-type and the other is H179R mut p53, which is supposed to have 10-fold less activity compared with the wild-type. My result showed ~7 fold decrease in the transactivity of p53 H179R, but the firefly luciferase values are similar between the two p53s, while it's the Renilla luciferase value of H179R p53 transfected wells increased ~7 fold. Does anyone have similar experience or some suggestions? Every suggestion would be helpful for me, and thanks very much!

Regards
Kevin


Generally speaking, the renilla is merely a transfection efficiency control. Your results suggest that your WT p53 vector has a lower transfection efficiency than your p53 H179R vector (higher renilla values=higher transfection efficiency). In that situation, though your luciferase values were similar, since your p53 mutant had a higher transfection efficiency and therefore more cells were generating luciferase, this means that each cell had less total activity than a p53 transfected cell. The similarity of the raw luciferase values is meaningless given the observed differences in renilla. Thus, it seems as though you have achieved results similar to the previous report you described. However, the 7 fold variation in efficiency is somewhat disconcerting and can likely be corrected with the measures below.

Is your H179R mutant in the same vector backbone as your wt p53? If not, it should be so as to account for promoter and flanking sequence differences that can affect expression.

Are you expressing similar amounts of protein for each transcription factor? Even a point mutation can greatly influence the production of a given protein such that a similarly transfected amount yields a much higher or lower expression level and influencing the outcome of your study. You should ensure that similar protein levels are observed for both the wt and mutant p53; however, given the disparity in your transfection efficiencies, this may not be the case as different numbers of cells will be producing wt vs. mutant protein. This highlights why you must attempt to correct your disparate transfection efficiencies.

Are you using equimolar amounts of H179R vs. wt p53? Using equal masses, say 1 ug of plasmid A vs. 1 ug of plasmid B is improper, yet frequently done. You should be transfecting the same number of molecules of plasmid A vs. plasmid B, which will vary unless the sizes of plasmid A and B are the same (using the same vector backbone for both can alleviate this problem). The difference between the equimolar amount and the total mass can then be completed with naked vector (assuming that you have naked vector available which does not influence your assay in any way).

Example:
50 ng/well of plasmid A + 100 ng/well reporter + 10 ng/well renilla reporter + 140 ng/well naked vector (total 300 ng/well)
vs.
70 ng/well of plasmid B (equimolar amount) + 100 ng/well reporter + 10 ng/well renilla reporter + 120 ng/well naked vector (total 300 ng/well)

One final possible caveat, is the activity you observed constitutive or do you treat with something to achieve activity? If the latter, you should ensure that your treatment does not affect renilla expression alone.

Edited by Dr Teeth, 15 March 2010 - 10:00 AM.


Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 neuronzy

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Posted 15 March 2010 - 07:12 PM

Thank you very much for your suggestions. I used the same backbone of p53 WT and H179R mutant for I generated the site mutation using the p53 WT expression vector(pcDNA3.1+) as a template by site-directed mutagenesis kit. Thus I think the similar amount of DNA(ugs) equals to equimolar of plasmids.

As for the transfectino efficiency, I used the same amount of DNA controled by A260 measurement, and the other mutated p53 which has similar activity to the p53 WT gave a similar Renilla results, only the H179R showed differences. Besides the transfection efficiency, is it possible that the p53 WT and H179R have different influence to the cell fate so that cells in wells that transfected with the WT p53 died more than that with the p53 H179R?

Finally, I did not treat the cells so the Renilla acvity did not be influenced.

#4 Dr Teeth

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Posted 16 March 2010 - 04:09 AM

Thank you very much for your suggestions. I used the same backbone of p53 WT and H179R mutant for I generated the site mutation using the p53 WT expression vector(pcDNA3.1+) as a template by site-directed mutagenesis kit. Thus I think the similar amount of DNA(ugs) equals to equimolar of plasmids.


Great!


As for the transfectino efficiency, I used the same amount of DNA controled by A260 measurement, and the other mutated p53 which has similar activity to the p53 WT gave a similar Renilla results, only the H179R showed differences. Besides the transfection efficiency, is it possible that the p53 WT and H179R have different influence to the cell fate so that cells in wells that transfected with the WT p53 died more than that with the p53 H179R?

Finally, I did not treat the cells so the Renilla acvity did not be influenced.


It is possible. Did you see more cell death? Have you tested with trypan blue, etc.? It could also be an effect on cell growth, I suppose.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley




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