title: Analysis of mRNA Translation Rate in Mouse Embryonic Stem Cells.
abstract: Regulation of gene expression is essential to enable embryonic stem cells (ESCs) to either self-renew or to differentiate. Translational regulation of mRNA plays a major role in regulating gene expression and has been shown to be important for ESC differentiation. Sucrose gradients can be used to separate mRNAs based on the number of associated ribosomes and this can be used as a readout of the rate of translation. Following centrifugation through a sucrose gradient, mRNAs can be recovered, purified, and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) to determine their ribosomal load in different cell states. Here, we describe how to differentiate mouse ESCs to Neural Precursor Cells (NPCs) and analyze the rate of translation of individual mRNAs by qRT-PCR following polysome fractionation.
authors.affiliation: Institute of Medical Biology, A*STAR, 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore.
authors.forename: Anisa B
authors.initials: AB
authors.lastname: Rahim
authors.affiliation: Institute of Medical Biology, A*STAR, 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore. leah.vardy@imb.a-star.edu.sg.|School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore. leah.vardy@imb.a-star.edu.sg.
authors.forename: Leah A
authors.initials: LA
authors.lastname: Vardy
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D011132:Polyribosomes / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
chemical_list: D012333:RNA, Messenger
keywords: Differentiation; Polysome; Quantitative real-time PCR; Stem cells; Translation