Luciferase assay-normalisation - (Apr/17/2012 )

Hi

I'm carrying out an experiment to look at gene-expression event and how this is changed when different concentrations of a particular protein are present. I've carried out a dual-luciferase assay for this with using different titrations of a Luciferase-dependent-gene. My luciferase and renila data show no particular pattern; with the SD error bars are slightly large due to difference in reading within the triplicate. Yet when I normalise the data (luci/renila) the pattern emerges and the error bar are reduced to a very acceptable range. Should I believe the pattern which emerged from the normalisation, taken into account it's the pattern I expected?

Thanks in advance

Hi madina,

The idea for using both renilla and firefly in the dual luciferase is so you can normalise your different conditions to the control luciferase.
I therefore say, you could trust your normalised results.

I assume you did 3 separate transfection experiments, not splitting your cells in 3 and simply use the same sample 3 times?

Each experiment is slightly different, with different cell numbers, pipetting mistakes, transfection efficiencies and incubation/growing times. You therefore do not necessarily get the same result for the control luciferase each time you repeat the experiment, to control for this you are supposed to normalise the data to your renilla.

Many thanks for your response...appreciate it

p.s. when I mentioned 3 times I was talking about a triplicate