Luciferase Reporter Assay - Data analysis (Jul/09/2007 )

Hi,
I have some questions about reporter assays. I’m using promegas dual luciferase reporter assay system. I’m new to this technique and I’m not sure how to interpret the result I get. First I just wander if it is good to compare the result to the negative sample (with "empty" vector), I think it is bad to do this if this is only supposed to be "backgroundsignal" or bias, but maybe this is some basal expression that I can compare with?

Secondly I think I have very low values. I have firefly readings of about 0,3-0,8 but renilla values from 100-1000 RLU. What does this low values mean? (or is it my renilla that is high?) If I do all the calculations I can see an induction by my transcription factor (two or four times) but in reality this is just an increase from 0,001(negative) to 0,002 or 0,004 (sample of interest). Is this at all reliable? How high readings do you usually get from your reporter vector?

Hoping for some help,
Matilda

The signal you get (RLU) depends on the assay you are running and the luminometer you are using. Never tried the Promega dual luciferase assay myself, but know that the renilla luciferase gives a much higher signal than the firefly luciferase. If your signal is to low, you can usually increase it by adjusting the gain of your luminometer.

Matilda,
I think that your firefly and renilla values sound a bit low. With the dual luciferase, we have renilla readings in the region of 100,000 to 1,000,000 and firefly a log or 2 lower depending on the reporter 1,000 to 10,000. Do you know how transfectable your cells are?

Having said that if your results are reproducible in several independent experiments, maybe that's fine. People normally show relative fold-inductions compared to the control sample. Maybe you should try and optimise your transfection efficiency or use more cells/DNA in the transfection.

Ceri