Validation and characterization of a viral antisense transcript with Northern blot analysis and qRT-PCR

The transcription of mammalian genomes has been shown to possess an intriguing complexity and numerous novel RNA molecules have been identified in the last 10-15 years. Viruses with large DNA genomes, especially herpesviruses, are also shown to generate many different RNA species and some of them may function as long non-coding RNAs. Viral genomes harbor several genes within close proximity to each other and can generate multigenic transcripts in addition to commonly observed antisense transcription. It is essential to study the biological roles of these transcripts aside from the protein-coding counterparts. A transcriptionally complex locus can be studied with a combination of methods. Generally, quantitative PCR assays are very commonly used for expression analyses of the transcripts of interest. Here an example from a gammaherpesvirus is discussed in more detail. A recently developed method, for the resolution of complicated transcriptomes for viral genomes, elucidated multiple antisense transcripts from ORF63-64 locus in murine gammaherpesvirus 68 (MHV68). In order to identify the roles of these new transcripts, quantitative PCR assays may not be enough alone and should be supported by alternative methods such as Northern blots. A more detailed transcriptional map of the locus of interest is useful to design experimental strategies and perform functional studies.