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Nonradioactive northern blot analysis to detect ebola virus minigenomic mRNA

Brauburger, Kristina LU ; Cressey, Tessa and Mühlberger, Elke (2017) In Methods in Molecular Biology 1628. p.143-159
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... (More)

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).

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author
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
keywords
Denaturing RNA gel electrophoresis, Digoxigenin-labeled RNA probe, Ebola virus, Filoviruses, Minigenome, mRNA detection, mRNA purification, Nonradioactive Northern hybridization, Northern blot, Vacuum blot
in
Methods in Molecular Biology
editor
Hoenen, Thomas; Groseth, Allison; and
volume
1628
pages
17 pages
publisher
Humana Press
external identifiers
  • scopus:85020234477
ISSN
10643745
DOI
10.1007/978-1-4939-7116-9_11
language
English
LU publication?
yes
id
3ff1b715-ce20-4c3b-941e-83cceea2f3df
date added to LUP
2017-06-30 09:58:29
date last changed
2018-01-07 12:10:01
@inbook{3ff1b715-ce20-4c3b-941e-83cceea2f3df,
  abstract     = {<p>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).</p>},
  author       = {Brauburger, Kristina and Cressey, Tessa and Mühlberger, Elke},
  editor       = {Hoenen, Thomas and Groseth, Allison},
  issn         = {10643745},
  keyword      = {Denaturing RNA gel electrophoresis,Digoxigenin-labeled RNA probe,Ebola virus,Filoviruses,Minigenome,mRNA detection,mRNA purification,Nonradioactive Northern hybridization,Northern blot,Vacuum blot},
  language     = {eng},
  pages        = {143--159},
  publisher    = {Humana Press},
  series       = {Methods in Molecular Biology},
  title        = {Nonradioactive northern blot analysis to detect ebola virus minigenomic mRNA},
  url          = {http://dx.doi.org/10.1007/978-1-4939-7116-9_11},
  volume       = {1628},
  year         = {2017},
}