title: Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization.
abstract: 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.
authors: Swammerdam Institute for Life Sciences, Universiteit van Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands. mstam@science.uva.nl.
authors: Max
authors: M
authors: Haring
authors: Sascha
authors: S
authors: Offermann
authors: Tanja
authors: T
authors: Danker
authors: Ina
authors: I
authors: Horst
authors: Christoph
authors: C
authors: Peterhansel
authors: Maike
authors: M
authors: Stam