We have quadruplicates for mitochondria copy number multiplex PCR. ND1 representing the mitochondrial DNA and RNAse P being our reference.

Equation for mitoCN= (2^(ND1 Ct-RNAseP Ct)) * 2

Question is- do we average the Ct's for the quadruplicates and then plug into the formula, or do we calculate the mitoCN for each well and then average the mitoCN for the 4 wells.

The two options obviously give a different mitoCN.

Thanks