**40**wrote:

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 ?

I believe the mentioning of the reference-normalised values is more of a finding and that it's not that they have scaled those values to the 0-15 range. That is, after they have subtracted the mean of 3 replicates for each gene of interest from the mean of the 5-panel reference, they made the observation that values ranged between 0-15. They state:

The mentioning of 'doubling' of RNA is purely related to the fact that each cycle difference in PCR reflects a duplication / copying of he original cycle's content.

In situations where a negative reference-normalised value is found, the values can be shifted by a specific factor in order to bring the range to have min=0. The eventual range for your particular data may eventually be 0-8 o 0-23, etc.

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Note, then, that these reference-normalised values are then used to produce the recurrence score (RS), which is on the range 0-100. As the method is proprietary, they do not go into the stats. However, I imagine that we're talking about a simple regression model here, with the 16-gene panel's reference-normalised values as predictors and some marker of relapse as outcome. Predictions from regression models are on the scale 0-1 (or 0-100%).

Kevin

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