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Luciferase assay problem

pGL3

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2 replies to this topic

#1 chonela

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Posted 04 July 2013 - 01:15 AM

Hi!

I am using Dual Luciferase System and the pGL3Basic vector to perform luciferase assays. After transfection with the pGL3 and Renilla-luciferase vectors, cells are treated with retinoic acid or the vehicle (DMSO).

I have noticed that cells treated with retinoic acid exhibited an increase in luciferase activity but the problem is that cells transfected with pGL3Basic presents high luciferase activity also. Could you please make any suggestion??

Thank you very much in advance!!

#2 Curtis

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Posted 07 July 2013 - 07:48 PM

I don't understand the problem. Can you elaborate?

#3 DrLeo

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Posted 08 July 2013 - 06:19 PM

First, you should use the same concentration of pGL3 and Renilla for both treated cells and controls.
Second, you should check the number of cells, should be similar between two groups.
Third, The Renilla signal should be higher than 10.000, regardless of Firefly signal, but higher is better, if it's under that value, you cannot use the data and should perform transfection again.
And, pGL3 basic vector had no promoter region of Luciferase, therefore the Luc activity is very weak.





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