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Northern Blot (non-radioactive)

Northern Blot analysis is a reliable hybridization technique frequently used for the detection of a specific RNA transcript (e.g. mRNA) within a complex mixture. Historically, mainly radioactively labeled hybridization probes have been used however, non-radioactive labels such as Digoxigenin, Biotin or near-infrared fluorescent dyes are attractive alternatives in terms of probe stability, handling convenience and improved safety profiles.[1-3]

Using RNA probes ("riboprobes") generated by in vitro transcription instead of double-stranded DNA probes results in significantly higher assay sensitivity due to increased riboprobe affinity to RNA target and a higher thermodynamic stability of RNA:RNA complex.[4-6] Riboprobes thus allow a reduced exposure time and more stringent washing conditions resulting in a lower background signal.

 

Selected References

[1] Miller et al. (2018) Near-infrared fluorescent northern blot. RNA 24(12):1871.
[2] O'Neill JW et al. (1994) Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. Biotechniques 17(5):870.
[3] Brauburger et al. (2017) Nonradioactive Northern Blot Analysis to Detect Ebola Virus Minigenomic mRNA. Methods in Molecular Biology 1628: DOI 10.1007/978-1-4939-7116-9_11.
[4] Melton et al. (1984) Efficient in vitro synthesis of biologicaly active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor. Nucleic Acids Research 12(18):7035.
[5] Srivastava et al. (2000) Analysis of RNA by Northern Blotting Using Riboprobes. The Nucleic Acid Protocols Handbook 38:249.
[6] Srivastava et al. (1991) Use of riboprobes for Northern blotting analysis. Biotechniques 11:584.