A laboratory practical illustrating the use of the ChIP-qPCR method in a robust model: Estrogen receptor alpha immunoprecipitation using Mcf-7 culture cells.

Chromatin immunoprecipitation followed by qPCR analysis (ChIP-qPCR) is a widely used technique to study gene expression. A large number of students in molecular biology and more generally in life sciences will be confronted with the use of this technique, which is quite difficult to set up and can lead to misinterpretation if not carefully managed. This article describes a four-session laboratory practical designed for Masters students to introduce this technique. During the practical, students work in pairs. They extract chromatin from MCF-7 culture cells stimulated or not by estrogens, then immunoprecipitate the transcription factor estrogen receptor alpha using an antibody directed against it. Students then measure the enrichment of promoter DNA target sequences from the chromatin that coprecipitates by qPCR. These are two estrogen responsive genes, pS2 (trefoil factor one) and PGR (the progesterone receptor). They learn how to analyze their ChIP-qPCR data by two methods: percent input and fold enrichment, and are taught about the interpretation limits of these two calculation methods. Thus, this practical is a good framework for an in-depth discussion of how this technique can be used to study gene expression and for raising awareness of the importance of careful interpretation of results.