normalization of dual luciferase assay
Posted 22 October 2010 - 01:04 PM
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
Posted 27 October 2010 - 07:30 AM
Posted 27 October 2010 - 10:16 AM
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.
Posted 28 October 2010 - 05:16 AM
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.
Posted 28 October 2010 - 06:48 AM
Yes you are right.
I understand it now.I dont know y it seemed to complicated to me.
Thank you very much.