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基本説明
Includes methods for state-of-the-art deep sequencing, miRNA-based shRNA-induced gene knockdown and qRT-PCR technologies. Protocols to identify and analyze the expression and function of RNAi and miRNA in stem cells.
Full Description
Recent stem cell research has revealed that miRNA and RNAi-mediated gene regulation is one of the vital determinates controlling the state of cell differentiation, with the small RNAs serving as key elements involved in regulatory network control of pluripotent cell fate determination. In RNAi and microRNA-Mediated Gene Regulation in Stem Cells: Methods, Protocols, and Applications, expert authors from laboratories across the globe contribute an accessible compendium of up-to-date, proven methods focused on the study of the titular topic. Divided into three sections, the book first gives a brief introduction to RNAi and miRNAs in stem cells, with a focus on the current status of research and future perspectives, then it continues with detailed methods and protocols for RNAi screening, transfection, and the knockdown of specific genes and pathways in several animal species, including humans and mice, concluding with a section on recently developed methods for identification of miRNAs, including a general protocol for preparation and analysis of miRNA libraries for deep sequencing, knock down of a specific gene using miRNA-based shRNA, and miRNA expression analysis using qRT-PCR. Written in the highly successful Methods in Molecular Biology™ series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes highlighting tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, RNAi and microRNA-Mediated Gene Regulation in Stem Cells: Methods, Protocols, and Applications serves as a valuable resource for scientists and aspiring graduate students interested in the intersection of RNAi, miRNA, and stem cell molecular biology and the exciting areas of medicine, including regenerative medicine, aging, cancer, and neurological disorders, that can be advanced through this expanding area of research.
Contents
RNAi in Stem Cells: Current Status and Future Perspectives.- A Brief Introduction to RNAi and MicroRNAs in Stem Cells.- RNA Interference.- Forward RNAi Screens in Human Stem Cells.- A Recessive Genetic Screen for Components of the RNA Interference Pathway in Mouse Embryonic Stem Cells.- Construction and Application of Random dsRNA Interference Library for Functional Genetic Screens in Embryonic Stem Cells.- Establishing Efficient siRNA Knockdown in Stem Cells Using Fluorescent Oligonucleotides.- Loss-of-Function Studies in Mouse Embryonic Stem Cells Using the pHYPER shRNA Plasmid Vector.- Regulation and/or Repression of Cholinergic Differentiation of Murine Embryonic Stem Cells Using RNAi Directed Against Transcription Factor L3/Lhx8.- Silencing of Rho-GDI? by RNAi Promotes the Differentiation of Neural Stem Cells.- RNAi Knockdown of Redox Signaling Protein Ape1 in the Differentiation of Mouse Embryonic Stem Cells.- Proteins Involved in Cell Migration from Glioblastoma Neurospheres Analyzed by Overexpression and siRNA-Mediated Knock-Down.- An Efficient Transfection Method for Mouse Embryonic Stem Cells.- Semi-quantitative Analysis of Transient Single-Cell Gene Expression in Embryonic Stem Cells by Femtoinjection.- MicroRNAs.- Preparation and Analysis of MicroRNA Libraries Using the Illumina Massively Parallel Sequencing Technology.- Assessing In Vivo MicroRNA Function in the Germline Stem Cells of the Drosophila Ovary.- Monitoring MicroRNA Expression During Embryonic Stem-Cell Differentiation Using Quantitative Real-Time PCR (qRT-PCR).- Engineering Human Mesenchymal Stem Cells to Release Adenosine Using miRNA Technology.- Efficient Gene Knockdowns in Mouse Embryonic Stem Cells Using MicroRNA-Based shRNAs.
