Look at this data:Īs you see, they don't include their control. Since fold change means compared to that of control, it assumes that all the controls are 1.ģ) Usually, you don't have to show the control group in your data presentation, because everyone knows that they are all 1, with no variation (no error bar). Let's say biological replicate #1 gave you 1.5 fold change, #2 gave you 2.0 fold change and #3 gave you 3.1 fold change. If they are significant, then just get the mean fold change and use it for the rest of the analysis.Ģ) Do the step 1 for each of biological replicate analysis. The key point is that there are two levels of statistical analysis involved: (A) Within each group (technical replicates), ( Between groups (when you compare the biological replicates).ġ) At the technical replicate level, make sure the ddCt numbers of your experimental sample is significantly different that the ddcts of the control group. I have been dealing with the same question for a long time. If anyone could give me any help on how to assess significance of the data (i have minitab and graphpad prism as stats/graphing programs) then it would be much appreciated. I would assume that the data is not normally distributed so it would have to be a non-parametric test, and i was assuming that the Man Whitney U test would be appropiate, but when i try and run this in minitab on the fold change values for a gene, it will not run, as the fold change for the control is 1 for every sample - could i therefore run the test on the CT values or DeltaDelta CT values instead? My problem now is how to i tell if the fold change is significant or not - which significant test should i use? I averaged the induced and normal fold change at the very end, to get the averga fold change for each gene under the induced and normal condition from which i used to draw graphs etc.
I had 2 reference genes of which i found the geometric mean, and then i worked out for each sampleĭelta CT (CT gene of interest - CT geomean ref gene)ĭelta Delta CT (Delta Ct induced - Delta CT normal) I then grew the cells to confluence, and set up an experiment either induced or normal/control in triplicate on three seperate days. I had 5 different dogs from which i took tissue. Hi, so i have performed QPCR on some tissue (induced and control).
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