miRNA transfection concentraion issue - Question about transfection reagent ratio (Jul/25/2008 )

Hi everybody,

I am currently doing a luciferase assay with pGL3 vector (400ng) and co-transfecting a mature miRNA of interest and a cel-miR negative control (both mimics are from Dharmacon). I use 1uL of Lipo2000 for transfection. I tried 4 different concentrations for the miRs - 0, 5, 150, 100 nM. The problem is that with the 50nM and 100nM transfection, even the negative control RLU/s ratio goes down!!

My question is...if you are transfecting different amounts of miRNA -5 vs 50 vs 100, do you increase the amount of Lipo2000 too from 1uL to 1.5uL to 2 uL or some ratio like that?

I would appreciate your insights and replies.

Thanks,
Micron

Hi everybody,

I am currently doing a luciferase assay with pGL3 vector (400ng) and co-transfecting a mature miRNA of interest and a cel-miR negative control (both mimics are from Dharmacon). I use 1uL of Lipo2000 for transfection. I tried 4 different concentrations for the miRs - 0, 5, 150, 100 nM. The problem is that with the 50nM and 100nM transfection, even the negative control RLU/s ratio goes down!!

My question is...if you are transfecting different amounts of miRNA -5 vs 50 vs 100, do you increase the amount of Lipo2000 too from 1uL to 1.5uL to 2 uL or some ratio like that?

I would appreciate your insights and replies.

Thanks,
Micron