Detection of viral microRNAs by Northern blot analysis.

ABSTRACT

microRNAs (miRNAs) of host and viral origin have been suggested to play important roles in the viral infectious cycle. The discovery of new viral miRNAs, and understanding how viral infection alters host miRNAs, has been greatly aided by Northern blot analysis. The Northern blot method is used to detect specific RNAs that have been separated by size and immobilized onto a membrane. This method can provide specific information regarding the size of a miRNA and possible precursor structures. Thus, it represents a valuable tool in the discovery and validation of new miRNAs. Viral infection can sometimes present special challenges to utilizing Northern blot analysis. These challenges may include low miRNA expression levels, high GC content, and abundant background signal from nonspecific RNA degradation fragments triggered by the stress of lytic infection. We present a protocol for small RNA Northern blot analysis that we have successfully used to detect viral miRNAs from cells undergoing lytic infection from members of the Herpes and Polyoma virus families. Included are optimization strategies and a protocol for using radiolabeled oligonucleotides to detect larger RNAs.