用于检测埃博拉病毒微型基因组mRNA的非放射性Northern印迹分析

Nonradioactive Northern Blot Analysis to Detect Ebola Virus Minigenomic mRNA.

作者信息

Brauburger Kristina, Cressey Tessa, Mühlberger Elke

机构信息

Department of Microbiology and National Emerging Infectious Diseases Laboratories, School of Medicine, Boston University, Boston, MA, USA.

Department of Biology, Lund University, Lund, Sweden.

出版信息

Methods Mol Biol. 2017;1628:143-159. doi: 10.1007/978-1-4939-7116-9_11.

DOI:10.1007/978-1-4939-7116-9_11

PMID:28573617

Abstract

In this chapter, we describe the detection of Ebola virus minigenomic mRNA using a nonradioactive Northern hybridization. This protocol comprises all steps beginning with the synthesis of a digoxigenin-labeled riboprobe, harvest of transcribed mRNA from cells transfected with the Ebola virus minigenome system, separation of mRNA species by denaturing RNA gel electrophoresis, transfer of the mRNA to nylon membranes by vacuum blotting, and finally the detection of minigenome-specific mRNA through hybridization with a labeled riboprobe directed against the reporter gene.This method allows the direct study of cis-acting regulatory regions as well as trans-acting factors involved in Ebola virus minigenome transcription compared to the indirect measurement of reporter protein activity that additionally reflects translational effects (see Chapter 6 in this book for details).

摘要

在本章中，我们描述了使用非放射性Northern杂交检测埃博拉病毒微型基因组mRNA的方法。该方案包括从合成地高辛标记的核糖探针开始的所有步骤，从用埃博拉病毒微型基因组系统转染的细胞中收获转录的mRNA，通过变性RNA凝胶电泳分离mRNA种类，通过真空印迹将mRNA转移到尼龙膜上，最后通过与针对报告基因的标记核糖探针杂交来检测微型基因组特异性mRNA。与额外反映翻译效应的报告蛋白活性的间接测量相比，该方法允许直接研究参与埃博拉病毒微型基因组转录的顺式作用调节区域以及反式作用因子（有关详细信息，请参阅本书第6章）。