Northern blotting is a gold standard technique for detecting specific RNAs. With this technique, RNA is denatured and separated by gel electrophoresis. The molecules are then transferred to a membrane that is incubated with labeled probes. Hybridization of complementary probes allows visualization of target RNA sequences (1). The related, hybridization-based technique RNase protection assays (RPA) improves the quantitation of RNA species, but data produced do not provide information about RNA size – a factor that can be important when studying transcript variants. Both approaches require minimal processing of RNA samples and are not dependent on reverse transcription or amplification
Hybridization-based RNA detection techniques require probes that are complementary to the RNA of interest and labeled for detection. For northern blots, hybridization probes can be labeled double-stranded DNA. Single-stranded hybridization probes reveal additional information with respect to the strandedness of RNA under study, which is not possible when dsDNA probes are used. Single stranded DNA oligos are often labeled with radioactive phosphate by using T4 Polynucleotide Kinase (NEB #M0201), or with easily detected chemical modification (e.g. biotin, fluorescent groups) during chemical synthesis. For RNase protection assays, single-stranded RNA probes are required. Strand-specific RNA probes can be generated and labeled by in vitro synthesis using radio-labeled nucleotides or by the incorporation of nucleotides labeled with detectable groups.
RT-PCR-based techniques are widely used for RNA detection, and when coupled qPCR approaches can have the advantage of being quantitative and moderately high throughput. While carefully designed experiments can reveal the structure of RNA (e.g. alternatively spliced mRNAs), information about the size, and integrity of the RNA under study is not reported. RT-qPCR is commonly used for studying mRNA expression.
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Press, 2007. 131-2.
- Rio, D. C., et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor
Laboratory Press, 2011, 77-87.