Your qPCR run is done and you have a column of Ct values. Here is how to turn them into fold changes with the ΔΔCt method, worked by hand, in a spreadsheet and in R, plus the mistakes that get qPCR figures sent back by reviewers.
If you test 20,000 genes at once, you’ll get 1,000 "significant" results by chance alone. This post explains the Multiple Testing Problem and how to use the p.adjust() function in R to calculate False Discovery Rates (FDR) and protect your results.