RNA sequencing is a technique widely used to identify and characterize gene expression patterns. We demonstrate that this method can be applied to screen expression profiles in mammary epithelial cells cultured in 3D, supported by a natural laminin-rich extracellular matrix, but requires several specific steps in the preparation of the RNA samples. Here we describe the use of RNA sequencing to analyze mRNA patterns in MCF10A human mammary epithelial cells cultured under 3D conditions in a laminin-rich extracellular matrix. We focus on our methods for total RNA extraction at early time points during the formation and maturation of 3D acinus structures in these cultures and provide examples of our results and downstream analysis.
Keywords: Acinus; Acinus formation; EMT; Epithelial–mesenchymal transition; Id-1; MCF10A cells; Mammary epithelial cells; Next generation sequencing; RNA sequencing.