To reduce any technical variation in your results Olink recommend that samples are randomized prior to analysis and that the results are intensity normalized.
When analyzing results from 2 or more studies analysed at different time points, and where samples can not be considered randomized between the studies, Olink recommend including between 8-16 bridging samples in each study. If possible, these reference samples should be representative for the entire study, for example include all study groups, and should be matrix-matched with other samples.
Using these bridging samples, reference sample normalization can be applied when merging previously normalized data sets.
Reference sample normalization is performed in the following way:
- Choose a reference project to normalize towards
- For each assay and project, calculate the pairwise difference for each of the overlapping samples with the reference plate
- Estimate the project- and assay-specific normalization factor by calculating the median for the pairwise differences calculated in step 2
- For each assay and project, add the plate- and assay-specific normalization factor from step 3 to each value, to normalize it to the reference plate chosen in step 1
NPX across plates for each protein including reference samples
NPX across plates for each protein after reference sample normalization
Data normalization FAQ
Sample randomization FAQ
How is the data pre-processed? FAQ
“Data normalization and standardization” white paper