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Luciferase assay - expected range of raw reading values

luciferase assay transfection

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#1 mbgene

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Posted 22 January 2012 - 12:31 PM

Hi there,

I am performing luciferase assays with Chroma-Glo Luciferase Assay System from Promega using the Mutlilabel Counter Victor3.
My mock transfected cells (as well as empty well measurement) always have low raw reading (~20), my positive control (transfected with a SV40-driven CBG68luc reporter gene) has raw reading of ~12,000.
My question now is in which range of values should I expect luminescent signals of my samples. My samples are HeLa cotransfectants, whereby the gene expression of one plasmid transactivates the second one, leading to luciferase expression. Raw values in which range would you consider as proper luminescent signals, and which ones would you rather consider as "background"?

Does anybody have an answer to this?

Thanks a lot!

#2 96well

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Posted 26 January 2012 - 05:42 AM

There are different ways to estimate the 'background', but in your case, I will do the following.
First, transfect HeLa cells with the luciferase plasmid plus the 'empty vector' corresponding to the transactivating plasmid, by doing so, the luciferase readings will be just background. Read various transfected wells (n=10-12), so you can have an accurate estimate of the standard deviation of your background (sB).

Now, just do your real assay with both luciferase and the right transactivation plasmid, again, for the beginning, I will use several wells. Calculate the mean of the Readings (uR).

Finally, you estimate the signal-to-noise ratio SNR as: SNR = uR / sB.
If your SNR is > 5, you are likely measuring proper signals. Having said that, it does not mean that the signal you measure has any biological significance, this is more matter of interpretation and common sense.

Here you can read some shortcomings of the luciferase assay

Edited by 96well, 26 January 2012 - 05:44 AM.

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