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The delta delta Ct method turns two Cq values per sample into a fold change in a minute of arithmetic. The two mistakes it hides take longer to spot: efficiency that is not quite 100%, which inflates your biggest results, and statistics run on fold changes instead of Cq values. This SOP runs from raw Cq to a fold change with a correct, asymmetric confidence interval.

If every measurement comes from a different animal, use ordinary two-way ANOVA. If the same animals are measured at every time point with nothing missing, use repeated measures ANOVA. If even one value is missing, use a mixed-effects model. Here is how to match the model to your design, what each mistake costs, and how to set each one up in GraphPad Prism.