To reduce technical variation between plates and studies Olink recommend that samples are randomized and that intensity normalization is performed before statistical analysis.
How do you normalize between plates of randomized samples?
For projects where samples are randomized, Olink apply intensity normalization with the plate median as the normalization factor. The intensity normalization adjusts the data to make the median value for each assay on each plate equal to the median for that of the other plates. This method assumes that the actual median of each plate is very similar which can be ensured by complete randomization of the samples. When complete randomization can be assumed, this is a robust and high performing normalization method.
Intensity normalization is performed in the following way:
For each assay, the overall median value for all samples and plates is calculated
For each plate and assay, the plate specific median value is calculated
For each assay, the plate specific median is subtracted from every sample of the plate, this centralizes the median to 0
For each assay, the overall median value is added to every sample in the project which equals centralizing to the overall median
NPX across plates for each protein
NPX across plates for each protein after intensity normalization
How can non-randomized projects be normalized?
If the samples have not or cannot be randomized Olink does not apply any extra normalization step. However, in a project with more than one plate of samples, we recommend performing a reference sample normalization with between 8-16 bridging samples run on all plates, to minimize technical variation. 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.
NOTE:Unlike intensity normalization this type of normalization is not included in the standard analysis service.
Reference sample normalization is performed in the following way:
Choose a reference plate to normalize towards
For each assay and plate, calculate the pairwise difference for each of the overlapping samples with the reference plate
Estimate the plate- and assay-specific normalization factor by calculating the median for the pairwise differences calculated in step 2
For each assay and plate, 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