real time PCR data analysis

Question

[Fatemeh]

Hello everyone, I have a question regarding qPCR data analysis. in my experimental set-up I have samples from 5 animals. From each animal I take 3 pieces of tissues and culture them separately. So, I have 3 replicates from each donor. Now, my question is that should I make average from replicates of one donor? I think the best is not to average. But the issue is that I need to somehow consider the donor impact in the analysis. I am using Graphpad Prism for data analysis, however, there is no option to consider the different donors in the analysis.

I would be grateful if you can help me with this.

[ATpoint]

I think the keyword to go after here is repeated measures https://www.graphpad.com/guides/prism/latest/statistics/repeated_measures.htm

[Fatemeh]

Thanks a lot for the link. The problem is that with this repeated measures, if you put the data from different donors in different rows, for example, then the graphs you will get out of it will be: one box plot for one donor. And this is not what I want. I want in the end to have : one box plot for all the donors.

[ATpoint]

I cannot guide on GraphPad. It's commercial software so better contact their support.

[Gordon Smyth]

Are you doing qPCR for one gene or for several genes?

[Fatemeh]

I have done for several genes, but I plot them in separate plots.

Answer 1

[Gordon Smyth]

If you are doing qPCR for several genes at once, then the analysis can be noticeably improved by using the limma package, see:

https://support.bioconductor.org/p/p134303/

limma allows you to specify repeat measurements on a donor. The use of the R environment, although requires it more expertise than GraphPad Prism, would also allow you to plot the results in any way that you choose.