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 |