Microarray Analysis of Embryonic Stem Cells and Differentiated Embryoid Bodies
By altering the cellular microenvironment and culture media composition, embryonic stem cells (ESCs) can be induced to differentiate in vitro into somatic cell types from the three primitive germ layers. ESC differentiation is regulated by an intricate se
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1. Introduction Culturing mouse ESCs (1) in vitro was a major scientific breakthrough that led to a series of significant biomedical research advances in transgenic (2) and knockout mouse models (3, 4) and provided valuable insight for the subsequent culture of human ESCs (5). Cultured ESCs have been used as a developmental model system to study gene and signaling networks that drive stem cells to differentiate into specific somatic cell types (e.g., cardiac myocytes (6)). Microarrays provide an opportunity to make unbiased genome-wide surveys to identify the transcriptional fingerprints of the gene networks that drive ESC differentiation (7) into somatic cell types. The use of genetically engineered
Sridar V. Chittur (ed.), Microarray Methods for Drug Discovery, Methods in Molecular Biology, vol. 632, DOI 10.1007/978-1-60761-663-4_3, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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Zambon and Barker
ESCs that express selectable transgenes (e.g., neomycin) (8) or fluorescent marker genes with tissue-specific promoters enables one to enrich and purify desired cell types. Such lines are well suited for genome-wide expression profiling. Several considerations should be made when choosing an ESC line, including the strain of mouse from which the line was derived from (considering that the ESC line will be used for genetargeted mutations and the generation of chimeric mice), whether or not the line requires coculture with mitotically inactive embryonic fibroblast feeder layers (i.e., culture of “feeder free” ESCs is less labor-intensive), and the potential for in vitro differentiation into desired cell types. This last consideration is supported by evidence of variability in the cardiogenic potential of various human (9) and mouse (8) ESC lines. It is important to note that variations in the culture conditions and genetic background of ESC lines can have a dramatic effect on gene expression signatures and should be taken into consideration when planning and interpreting expression profiles of ESCs and ESC-derived cells (10). A variety of microarray platforms and sample preparations have been described (for review (11)). The most commonly used array platforms available today are Affymetrix, Agilent, and Illumina microarrays. When selecting an array it is important to keep in mind that while different array suppliers may detect the same RNA transcript, the exact probe sequences used on each array can be quite different and located on different exons within a transcript. As a result, it can be problematic to directly compare data from similar samples that were run on different kinds of arrays. We recommend that array users use the same array across multiple data sets to facilitate future meta-analyses. While we discuss the classical sample preparation for Affymetrix microarrays by in vitro transcription, in many cases, it is not possible to obtain the amounts of RNA required for the described protocol. Numerous commercial sample preparation kits available also work quite well, including Affyme
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