Usually renilla is a constant amount in an assay, but in my case I used 100ng of PGL3 and varied the amount of renilla in the cells. If I normalise the data by dividing the PGL3 by renilla as usual it shows the result that as there is less renilla there is more luminescence.
However if I divide renilla by PGL3 it shows that as the level of renilla increases so does the level of luminescence, which is more in line with the expected results.
I'm just wondering if this is completely wrong as in my controls it is showing a higher RLR than in my samples so I am doubting myself.
Thank you for your time
What is an RLR? Dividing by PGL3 sounds logical in the experiment that you have mentioned, since PGL3 should be a constant value, if I am not mistaken.
sorry, RLR is relative luminescence ratio. I have posted a quick summary of my results in both formats below if that helps. I just can't seem to understand the results at all.
I think you need consider use psiCHECK2 vector. it has Renilla and Firefly luciferase expression at the same time. Renilla luciferase activity was normalized to Firefly luciferase expression.
you can go to look my paper PMID:24401233. (Supplemental Material Online). or you can check the vector information online.
In my experiment, renilla is not a constant amount. it is a report gene.
psiCHECK2 vector have Renilla and Firefly luciferase expression at the same time. between them, I insert one seq as a target. Firefly luciferase is upstream, so it always expressed after your transfection. if the target be blocked, the Renilla luciferase will not express in cells.
So, I don't think renilla luciferase is a constant amount in our system. you need to find other one normalized it.
I know maybe my experiment system is not fit your experiment. you can read the link:
http://www.promega.com/~/media/Files/Resources/Cell%20Notes/cn017/Normalizing%20Genetic%20Reporter%20Assays.ashx
it will help you find the right one.
Hope this helps, good luck
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