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low signals of luciferases in PPARg transfection - (Jul/08/2008 )

I have transfected NF-kB or PPARg-Luc plasmid together with p-RLTK as internal control in HT29 cells.

Using GeneJammer, I have read very good firefly and renilla signals (like 1000-20000 RLU) from NF-kB tranfected cells after induction. But I am getting very low signal in PPARg cases (only 50-100 RLU in firefly and 150-700 RLU in renilla) consistently in HT29 cells. I cant say this is because of the transfection efficiency, as NF-kB is working well in the same cell line.

Importantly, the PPARg has given good signals in HT1080 cells using the same reagents and conditons.

What may be the problems of PPARg in HT29 cells? Just because of change of plasmid cause variations in transfection efficiency in such a great extent? Aren't these signals are just basal background signals? What does very low firefly signal reflect??

Thank you.

Thapa

-Thapa-

Always do a optimization titration (reagent to DNA ratio) for every cell line and plasmid because the optimal ratio can vary from condition to condition. I do the same with FugeneHD which I prefer over GeneJammer or others because of the lower cytotoxic. and off-site effects.
Maybe the PPARg is somehow cytotoxic to your cell line?





QUOTE (Thapa @ Jul 9 2008, 06:45 AM)
I have transfected NF-kB or PPARg-Luc plasmid together with p-RLTK as internal control in HT29 cells.

Using GeneJammer, I have read very good firefly and renilla signals (like 1000-20000 RLU) from NF-kB tranfected cells after induction. But I am getting very low signal in PPARg cases (only 50-100 RLU in firefly and 150-700 RLU in renilla) consistently in HT29 cells. I cant say this is because of the transfection efficiency, as NF-kB is working well in the same cell line.

Importantly, the PPARg has given good signals in HT1080 cells using the same reagents and conditons.

What may be the problems of PPARg in HT29 cells? Just because of change of plasmid cause variations in transfection efficiency in such a great extent? Aren't these signals are just basal background signals? What does very low firefly signal reflect??

Thank you.

Thapa

-Senior_Scientist-

Many thanks Senior Scientist. Currently I am trying to optimize using the same reagent. Lets see if it works..

QUOTE (Senior_Scientist @ Jul 15 2008, 05:12 AM)
Always do a optimization titration (reagent to DNA ratio) for every cell line and plasmid because the optimal ratio can vary from condition to condition. I do the same with FugeneHD which I prefer over GeneJammer or others because of the lower cytotoxic. and off-site effects.
Maybe the PPARg is somehow cytotoxic to your cell line?



-Thapa-