用于原位杂交实验的地高辛标记核糖探针的制备

Q1 Agricultural and Biological Sciences

Current protocols in mouse biology Pub Date : 2020-05-21 DOI:10.1002/cpmo.74

Kristen S. Barratt, Ruth M. Arkell

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引用次数: 6

摘要

在完整的组织或有机体中可视化基因表达的实验是研究基因功能的基础。这些实验被称为原位杂交，需要产生一种核蛋白探针，这是一种与特定基因相对应的标记反义RNA。通过原位杂交可视化基因表达最常用的系统是将地高辛标签掺入体外转录RNA探针中。将核糖核酸探针与目标mRNA杂交后，可以通过与碱性磷酸酶偶联的高亲和力α-地高辛抗体检测其位置。本文介绍了从模板DNA(质粒或PCR扩增子)体外转录地高辛标记的核糖探针的设计和生产。这些核糖探针适用于组织和全贴装原位杂交方案。©2020 by John Wiley &基本方案1:质粒衍生的核糖探针;备选方案:pcr衍生的核糖探针;基本方案2:用DIG标记合成核糖探针

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Production of Digoxigenin-Labeled Riboprobes for In Situ Hybridization Experiments

Experiments that visualize gene expression in intact tissues or organisms are fundamental to studies of gene function. These experiments, called in situ hybridization, require the production of a riboprobe, which is a labeled antisense RNA corresponding to a particular gene. The most commonly used system for visualizing gene expression via in situ hybridization is the incorporation of a digoxigenin label into an in vitro−transcribed RNA probe. After hybridization of the riboprobe to a target mRNA, its location can be detected via a high-affinity α-digoxigenin antibody conjugated to an alkaline-phosphatase enzyme. The article describes the design and production of digoxigenin-labeled riboprobes transcribed in vitro from template DNA (either plasmid or PCR amplicon). These riboprobes are suitable for use in tissue and whole-mount in situ hybridization protocols. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Plasmid-derived riboprobes

Alternate Protocol: PCR-derived riboprobes

Basic Protocol 2: Riboprobe synthesis with DIG label

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来源期刊

Current protocols in mouse biology Agricultural and Biological Sciences-Animal Science and Zoology

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet, all too often, nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. The aim of Current Protocols in Mouse Biology is to provide the clearest, most detailed and reliable step-by-step instructions for protocols involving the use of mice in biomedical research. Written by experts in the field and extensively edited to our exacting standards, the protocols include all of the information necessary to complete an experiment in the laboratory—introduction, materials lists with supplier information, detailed step-by-step procedures with helpful annotations, recipes for reagents and solutions, illustrative figures and information-packed tables. Each article also provides invaluable discussions of background information, applications of the methods, important assumptions, key parameters, time considerations, and tips to help avoid common pitfalls and troubleshoot experiments. Furthermore, Current Protocols in Mouse Biology content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Mouse Biology brings together resources in mouse biology and genetics and provides a mouse protocol resource that covers all aspects of mouse biology. Current Protocols in Mouse Biology also permits optimization of mouse model usage, which is significantly impacted by both cost and ethical constraints. Optimal and standardized mouse protocols ultimately reduce experimental variability and reduce the number of animals used in mouse experiments.