Hello, I recently performed a high throughput qPCR experiment using the Biomark Fluidigm system. (Allows 96 samples x96 genes per chip.) The software spits out the CT values, delta ct, ddct values and the fold change. As to not blindly accept the software I started investigating the math that was done and confirmed with technical support. My experimental design is comparing out knock out mouse model to control animals.
The software does the following: Step 1: For all of the control samples (biological replicates, not technical) it averages the Ct value for the housekeeping gene and averages the Ct value for the gene of interest (GOI) Step 2: It finds the delta Ct value for the controls by= (average control GOI- average control Housekeeping) Step 3: Finds the delta Ct value for each individual sample by = (individual KO sample - average control MBP) Step 4: Delta delta Ct= step 3 value- step 2 value Step 5: 2^-ddct
This is not quite how I learned how to do the delta delta CT method. Does this method have any validity?