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Transient transfection efficiency too good? - Luriferase reporter assay (Jun/23/2010 )

Hi all,

I recently encounter a problem which i am not sure if it is normal. I have often heard about low transfection efficiency but have you come across cases where the transfection efficiency is too high?

When i first did my transient transfection with a luciferase reporter plasmid, i got a baseline value of RLU at about 20,000 - 30,000. The value goes up following treatment that stimulates the expression of the plasmid, and goes down with antagonist treatment, just as i expected.

But when i tried it for the second time, i got a baseline value of RLU at about 150,000 (about 5 - 7 x higher than before). The RLU value didn't get any higher following stimulation, but was reduced with antagonist to about 60,000 RLU.

Is it possible that when the transfection efficiency is too good (judging from the high basal value) that the co-regulators got mopped up altogether, and hence further stimulation given will not be able to increase the transcription of the plasmid? I know that the machine can read up to 500,000 RLU at least, so i don't think i have reached the plateau when i don't see any higher RLU value following stimulation.

I am planning to half the amount of plasmid DNA the next time i do it, meanwhile, would really appreciate your feedback...

Thanks!

-WYF-

WYF on Jun 23 2010, 04:34 PM said:

Hi all,

I recently encounter a problem which i am not sure if it is normal. I have often heard about low transfection efficiency but have you come across cases where the transfection efficiency is too high?

When i first did my transient transfection with a luciferase reporter plasmid, i got a baseline value of RLU at about 20,000 - 30,000. The value goes up following treatment that stimulates the expression of the plasmid, and goes down with antagonist treatment, just as i expected.

But when i tried it for the second time, i got a baseline value of RLU at about 150,000 (about 5 - 7 x higher than before). The RLU value didn't get any higher following stimulation, but was reduced with antagonist to about 60,000 RLU.

Is it possible that when the transfection efficiency is too good (judging from the high basal value) that the co-regulators got mopped up altogether, and hence further stimulation given will not be able to increase the transcription of the plasmid? I know that the machine can read up to 500,000 RLU at least, so i don't think i have reached the plateau when i don't see any higher RLU value following stimulation.

I am planning to half the amount of plasmid DNA the next time i do it, meanwhile, would really appreciate your feedback...

Thanks!


Yeah, sometimes If you use too much reporter plasmid you can mask the results. Ive seen that very often with Gal4-Luc assays, where if you use too much ProteinX-Gal4 the induction is so high that you cannot see any further increase.
I would use less reporter plasmid

-laurequillo-

Thanks!



Yeah, sometimes If you use too much reporter plasmid you can mask the results. Ive seen that very often with Gal4-Luc assays, where if you use too much ProteinX-Gal4 the induction is so high that you cannot see any further increase.
I would use less reporter plasmid


Thanks a lot for sharing with me laurequillo! I am almost at the brink of insanity as no one has ever done this before in my lab and i have no one to turn to for advice...Will try that again next week!

-WYF-