Hello, I would appreciate getting help to do appropriate statistical analyses on my qPCR data in GraphPad Prism for the sake of the statistical tools and graphing power.
I had already done the delta delta Ct analysis in Excel. I am dissatisfied because Excel doesn't offer much statistical power or good data visualization. Additionally, looking at the 2^-deltadeltaCt does not follow a Gaussian distribution, so nonparametric tests are required.
My experimental design is as follows:
3 technical replicates were done per sample
2 biological replicates were done per condition
I have 1 control (untreated sample) and 5 additional conditions
I have 2 genes of interest and am normalizing to GAPDH
So, for example, for each gene of interest tested (as well as GAPDH), I had two wells of iPSC untreated, isolated the RNA from each, then used the RNA from each well to do 3 PCRs for 1 GOI.
If I just try giving graphpad the 2^-deltadeltaCt to compare and analyze, then it will only see a sample size of 2 for each condition and there will be no statistical significance found between conditions.
How would you recommend I analyze this data in Graphpad?
Since I am still relatively new to GraphPad and not a statistician, it would be appreciated if more of a step-by-step solution is offered.
Thank you.
Dear Draven,
I would say that 2 replicates per group is insufficient, you should have at least 4-5 replicates for each condition to get any more or less reliable result.
In order to play with your data and try to perform statistical analysis, you may treat every technical replicate as an individual sample, thus having 6 samples per group (but as you understand, this approach is incorrect and will not give you publishable results).
As for the step-by-step description of the analysis, I would recommend my answer to a similar question: https://www.researchgate.net/post/Qpcr-statistical-analysis/1
But as you have more than two groups, for your data you will have to use either ANOVA (+ post hoc Tukey or Sidak - if the distribution is normal) or Kruskell-Wallis (+ post hoc Mann-Whitney - if the distribution is non-normal) - instead of t-test or Mann-Whitney.
Here is a wonderful video with an explanation of how to perform one-way ANOVA analysis:
https://www.youtube.com/watch?v=kpGDAetOrFo
If you wish, you can send me your dataset in Excel, and I'll send you back all the calculations and GraphPad Prism analysis.
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