Oncotype DX 21-gene panel is a popular panel for breast-cancer recurrent prediction and has been cited ~thousands times (See DOI: 10.1056/NEJMoa041588 ). What confused me a lot is the first step of expression level normalization for 16 cancer-related genes. Almost every literature wrote:
For each sample, normalised expression measurements are calculated as the mean cycle threshold (CT) for the 5 reference genes minus the mean CT of triplicate measurements for each individual gene. Normalized expression measurements are scaled from 0 to 15 units, where 1 unit reflects an ~2-fold change in RNA quantity
Why 0~15? Suppose the mean CT values for 5 reference genes is 28, and the CT for target gene MKI67 is 32. How can I calculate the normalized expression level for gene MKI67 ?