Nonradioactive Northern Blot Analysis to Detect Ebola Virus Minigenomic mRNA

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).