# How to interpret dual luciferase assay result (RLU)?

I performed a dual luciferase assay using pGL3 firefly luc as experimental reporter and pRL-TK renilla luc as internal control (Promega assay kit). I used a manual luminometer to analyze my sample. After that, I got following result:

Sample        RLU (LARII)                   RLU (stop & glo)

A               1978                               902

B               1702                               1101

C               1952                               980

As I am doing it for the first time, I cannot understand the result. I want to know, how can I interpret this result? There is no unit. RLU stands for relative luminescence unit; but what it actually means? What is the method to compare the results of different samples? How can I understand expression efficiency? I have read some papers but unfortunately my concept is not being clear.

Where can I find useful information and guidance about it?

Relative luminometer units (RLU) have no unit. They are the result of the luciferase reaction and proportional to the expression of the luciferase gene, meaning the higher the numbers are, the higher the expression is.

To analyze these numbers, you usually use a control, to which your samples are then relative to. So expression from your luciferase vector is 10x higher in the treated sample compared to the control for example.

As the RLU has no unit, it is hard to compare measurements directly. To overcome this problem, you can use a second luciferase reaction (the renilla luciferase) to be able to normalize between samples and to correct for transfection efficiency, but still, your values are only relative.

They are influenced but the kit you use for the measurement, the machine you use (so always use the same machine) and so on.

To normalize the values, you divide each firefly luciferase value by the value for the renilla in the same well. Ideally alls renilla measurements are the same (as you transfected the same amount of plasmid in each well, have the same number of cells etc., but if not, this corrects exactly this problem). You cannot calculate the transfection efficiency from this control.

To calculate the relative expression ratios between your sample B and the respective control A, you set the control to 1 by dividing A/A=1 (for example: A=10, 10/10=1) The relative expression of B is then B/A= relative expression (for example: B=5, relative expression: B/A= 5/10=0.5). In the example the relative expression of sample B would be half the one of the control.

• Thank you for your answere. It is helpful; although I am not completely clear. I have used renilla luc here to normalize the expression which is indicated here as RLU (stop & glo) values. And sample A indicates the control. Now, how can I compare my results? Can you give me one example please? Will I have to make a ratio of the two values (firefly RLU: renilla RLU)? And what does the ratio mean? How can I calculate the transfection efficiency? Please help me to know this. @Chris Mar 27, 2017 at 6:35
• @MRahman Is this clearer now?
– Chris
Mar 27, 2017 at 14:13