4 replies to this topic

[ariaz]

Hi
I am new to luciferase assays. I am finding it difficult how to normalize data.
I have psicheck2 vector which has both renilla and firefly. I ligated my gene of interest into 3'UTR of renilla gene.I need to see the reduction in renilla level, While firefly is working as control.
After performing luc assay i got readings using 20/20 glomax luminometer. First reading was of firefly and second was of renilla.
Can any one please help me out that how i can i normalize my data or how can i calculate the reduction in renilla level.
Thanks in advance

[96well]

In your case, for each well I will calculate the ration Renilla/Firefly and then compare those ratios among your experimental groups. You probably get lost because it is usally the Firefly plasmid being modified and the Renilla one used as normalizator, so the glomax luminometer would give the calculations as Firefly/Renilla.  In your case, you have to do Renilla/Firefly

[ariaz]

Thanks alot:
I was going to do in the same way but some one said that i should first calculate ratio Renilla/Firefly and then devide the renilla values with the averages of these ratios (Renilla/renilla firefly reatio average). I dont know it is right or not. What do you say.
Thanks again

96well, on 27 October 2010 - 07:30 AM, said:

In your case, for each well I will calculate the ration Renilla/Firefly and then compare those ratios among your experimental groups. You probably get lost because it is usally the Firefly plasmid being modified and the Renilla one used as normalizator, so the glomax luminometer would give the calculations as Firefly/Renilla.  In your case, you have to do Renilla/Firefly

[96well]

Imagine you have a single well in your experiment, so a single end-point

Renilla =  150
Firefly = 8000

R/F = 0.01875

average R/F = 0.01875

R/(averageR/F) = 8000

Is like if you divide Renilla/Renilla/Firefly: you erase Renilla values and obtain just the Firefly, I don't see how this could be useful.

[ariaz]

96 well,
Yes you are right.    
I understand it now.I dont know y it seemed to complicated to me.
Thank you very much.