Steps in the NPX generation described in equation form, where i refers to a specific assay, j refers to a sample and ExtNPX defines an extension normalized NPX value
1. ExtNPX_i, j = log2(counts(sample_jAssay_i)/counts (ExtCtrl_j))
a. Relate counts to known standard (Extension control)
b. For all assays and all samples, including negative controls, Plate Controls, and Sample controls.
c. Log2 transformation gives more normally distributed data
2. NPX_i, j = ExtNPX_i, j-median(ExtNPX(Plate Controls_i))
a. Perform plate standardization
b. For all assays and per plate of samples
3. NPX_Intnorm_i,j = (NPX_i,j – plate median(NPX_i)) + global median(NPX_i)
a. Between plate normalization (Optional but default for multi plate projects)
b. For each assay, for all plates in project.