Pre-processing of the data is achieved using our Olink® NPX Manager software – see the product page for more information.
Data pre-processing includes QC-steps and the generation of NPX values through a three-step normalization procedure.
NPX is derived from the Ct values obtained from the qPCR using the following equations:
CtAnalyte – CtExtension Control = dCtAnalyte
dCtAnalyte – dCtInter-plate Control = ddCtAnalyte
Adjustment against a correction factor:
Correction factor – ddCtAnalyte = NPXAnalyte
NPX is a relative quantification unit logarithmically related to protein concentration. Even if two different proteins have the same NPX values, their absolute concentrations may differ. NPX should be compared for each assay separately between samples within a run. NPX should not be compared between runs without proper inter-plate normalization due to the risk of falsely interpreting shifts in median between runs as a biological difference.
Olink recommends one of two normalization methods depending on the study design:
For randomized studies, we recommend intensity normalization. Intensity normalization can also be used when combining studies from different sources as long as the sample distribution can be considered comparable between the data sets.
For non-randomized studies, Olink recommends reference sample normalization. Running reference samples on all
plates is a good strategy to minimize technical variation.
When applied correctly, both intensity normalization and reference sample normalization can increase the power in a given study by reducing technical variation.
For more information see our white paper, “Data normalization and standardization“.