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. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):152-160, 2017.
Cancer Research Center of Toulouse-UMR1037 Inserm, Université Toulouse III Paul Sabatier, Oncopole de Toulouse, 31037 Toulouse Cedex 1, France.
Eric
E
Lacazette
D001665:Binding Sites; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007753:Laboratories; D061986:MCF-7 Cells; D017145:Models, Educational; D008967:Molecular Biology / Q000193:education*; D018794:Problem-Based Learning / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D011980:Receptors, Progesterone / Q000235:genetics
C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D011980:Receptors, Progesterone
ChIP-qPCR; epigenetics; gene expression; immunoprecipitation; molecular biology