Hi Giulia Lencioni . Your approach to calculate the fold change activity of the Wnt protein appears generally correct, but let's break it down step by step: Calculate the Mean for Each Condition: Yes, this is correct. You should calculate the mean for each condition across the three replicates. This will give you the average Firefly and Renilla luciferase activity for each condition (IND, NEG, POS, and NTC). Subtract the NTC Mean Value: Yes, this is generally correct. Subtracting the NTC mean value from the IND, NEG, and POS mean values helps to remove background noise. This is an important step because it corrects for any non-specific activity or autofluorescence in the cells. Calculate the Ratio of Firefly to Renilla Signals: Yes, this is correct. You should calculate the ratio of Firefly to Renilla signals for each condition (except for NTC). The Renilla luciferase activity serves as an internal control to account for variation in transfection efficiency and cell viability. The ratio normalizes the Firefly luciferase signal, which represents the activity of the Wnt pathway. Calculate the Fold Change: Yes, the fold change in Wnt activity is typically calculated as the ratio of the normalized Firefly signal in the IND condition (Wnt pathway induced) to the normalized Firefly signal in the NEG condition (Wnt pathway not induced). This gives you the fold induction of the Wnt pathway due to the treatment.

Regarding the POS control, its primary use is to provide a benchmark for maximum possible luciferase activity and to ensure that the assay components are working properly. It's not typically involved in the calculation of fold change for your experimental conditions, unless you're interested in comparing your experimental induction to this maximum possible level of induction.

Share

Thank you, Saman Behboodi Tanourlouee, a lot; you were very clear.

Now I was also wondering if it could be more correct to:

1) firstly, calculate the ratio Firefly/Renilla for each condition (POS, NEG, IND and NTC)

2) then do the mean of the triplicates

3) remove the NTC signal

4) calculate the fold change in activity as IND/POS ratio

Mathematically it should be the same, but I wanted to know which procedure is more correct from a theoretical point of view.

I really appreciate any help you can provide.

Dear Giulia Lencioni

Your alternative approach is also a valid method for analyzing luciferase reporter assay data, and the choice between the two methods could be influenced by the specifics of your experiment and the data distribution. Calculating the Firefly/Renilla ratio for each condition first: This method would account for any variability in transfection efficiency and cell viability on an individual sample basis, rather than averaging these effects across triplicates first. If the variability within your triplicates is high, this might be a preferable approach. Calculating the mean of the triplicates: This is standard practice for any experiment performed in replicate. It will provide a more robust estimate of the true value and will help to minimize the impact of outliers or minor experimental errors Removing the NTC signal: This is generally correct and helps to remove the background noise. Calculating the fold change in activity as IND/POS ratio: This step is not typically done, because POS is usually an overstimulation condition to serve as a benchmark for assay functionality. However, it might make sense in a situation where you are interested in how the experimental condition (IND) compares to the maximum possible level of induction (POS).

In summary, both your initial and alternative approaches are theoretically sound. The choice between them would likely depend on the specifics of your experiment and the distribution of your data. Some researchers might prefer to calculate ratios first to account for variability within replicates, while others might prefer to calculate means first to get a more robust estimate of each condition's true value.

As always in science, it's best to ensure that your chosen method aligns with the question you are trying to answer, is justifiable, and is transparently reported. That way, anyone reading your results can understand your analytical approach.

Keep asking great questions!

Thank you a lot Saman Behboodi Tanourlouee !! You were so helpful!