Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization.
BACKGROUND
Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation. A very useful technique to study these processes is chromatin immunoprecipitation (ChIP). ChIP is widely used for a few model systems, including Arabidopsis, but establishment of the technique for other organisms is still remarkably challenging. Furthermore, quantitative analysis of the precipitated material and normalization of the data is often underestimated, negatively affecting data quality.


RESULTS
We developed a robust ChIP protocol, using maize (Zea mays) as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR) is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches.


CONCLUSIONS
Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR) is the best method to analyze the precipitates, and present comprehensive insights into data normalization.
Haring
Max
M
Swammerdam Institute for Life Sciences, Universiteit van Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands. mstam@science.uva.nl.
Offermann
Sascha
S
Danker
Tanja
T
Horst
Ina
I
Peterhansel
Christoph
C
Stam
Maike
M