Basically, you can consider RLU as light intensity, which is a number assigned by the instrument---luminometer to your sample.  It has NO UNIT.  LAR II measure luciferase activity in your sample, which represents the activity of promoter etc. the things you want to determine.  "Stop & glo" measure the Renilla luciferase activity, which should be similar as same amount of Renilla luciferase plasmid is supposed to be used.  If the "Stop & glo" values have a big variation, which means your transfection efficiency are not same.  You can calculate ratio of LAR II / "Stop&Glo" and use  the ratio to compare different samples.  Hope this help. Good luck!

Hi

As mentioned by Wei Li, renilla activity gives you a measure of transfection.  Therefore you have to always normalize the renilla values with your firefly values (LAR). For comparison you have to use firefly/renilla activity ratio. 

One thing I would like to say about yours results. The F/R of you pGL3  vector only is higher or almost equal to your samples. To put it another way, there is no difference between the luciferase activity of your empty vector and your desired promoters. pGL3 does give leaky expression because of some cryptic promoter sites. I would advise you to revisit whether your promoters are properly cloned.

Hello...

I was reading your question and felt that maybe you could help me with my luciferase assay.

I'm doing luciferase assay in transfected cells with two differents alleles of a polymorphism. Both alleles are cotransfected with my microRNA from interest and a microRNA negative control. One allele has "seed site" from this microRNA and the other allele don't.

I found differente expression of my gene comparing the alleles + miRNA, but what I can not understand why my negative control miRNA is keeping different in the alleles comparison.

It should be the same gene expression in both alleles cotransfected with negative control, right? 

Anyone does have an idea what could be happening?

Thanks a lot!

I have a question. I see that this is a qualifiable assay, is the gene being transcribed/translated, but how is this quantifiable for anything other than substrate (assuming oxygen and ATP are not limiting factors)?

You better to take duplicate values of LARII and Stop values for each sample. Separately calculate the LARII/Stop value to normalization and finally get the mean value of that.

I think you may be not got the properly result. I usually got result like 10000-999999, never got result as 900-1999.

I normally take quadruplates for each condition i.e. control and treatment. From the quadruplates, I only take three similar values for luciferase and renilla and make the calculation . I calculate the ratio by dividing LARII/stopglo.

Hope this helps.

All the best.

My control value is 42.00 ± 0.602 and test value is 48.68 ± 0.450. How do I interpret it?