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Table 2 ChIP-QPCR normalization strategies

From: Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization

Normalization strategy Normalization method Points of interest
No normalization ChIP data is not normalized, equal amounts of input chromatin are used in different experiments Differences in chromatin purity may cause differences in ChIP signal
Background subtraction The no-antibody signal is subtracted from the ChIP signal Background signal levels in the NoAb control samples may be different from those in the ChIP samples, depending on the used primer set, chromatin purity and sample handling. The normalization will be strongly influenced by fluctuations in the background signal
Fold enrichment Normalization to background signal See Background subtraction
% of input Normalization to the amount of input chromatin Differences in sample handling between input and ChIP samples affect the normalization
Relative to control genes Normalization to the ChIP signal obtained at a control sequence Control sequences need to be developed. Control ChIP signals may differ between different tissues and developmental stages
Relative to nucleosome density Normalization to the ChIP signal obtained with an antibody for unmodified histone protein ChIP signals obtained with different antibodies are difficult to compare quantitatively. Regions with low nucleosome density can yield incorrect normalization