Included are optimization strategies and a protocol for using radiolabeled oligonucleotides to detect larger RNAs. Abstract This unit describes the processes of extracting RNA from tissues and analyzing it by northern blot hybridization to probe the expression of a particular gene at the transcriptional level. 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. 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. Viral infection can sometimes present special challenges to utilizing Northern blot analysis. Thus, it represents a valuable tool in the discovery and validation of new miRNAs. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting) unfractionated RNA is immobilized by slot or dot blotting. This method can provide specific information regarding the size of a miRNA and possible precursor structures. Abstract Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. All of these can be used to confirm results from microarray analysis and differential display experiments. The Northern blot method is used to detect specific RNAs that have been separated by size and immobilized onto a membrane. Description: Here, we review three popular RNA detection/quantitation methods: Northern blot analysis, Ribonuclease Protection Assays (RPAs) and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The discovery of new viral miRNAs, and understanding how viral infection alters host miRNAs, has been greatly aided by Northern blot analysis. MicroRNAs (miRNAs) of host and viral origin have been suggested to play important roles in the viral infectious cycle.
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