Abstract
RNA gel blots (often referred to as Northern gel blots) are frequently used in the analysis of gene expression, for example when investigating specificity of gene expression, quantification of transcription, and analysis of RNA processing. Electrophoresis of RNA through agarose gels for blotting requires complete denaturation of the RNA. A number of denaturants have been used, including glyoxal and dimethyl sulfoxide (1), methylmercuric hydroxide (2), and formaldehyde (3). The following protocol described by Fourney et al. (4), uses formaldehyde as the denaturant but at a lower concentration than previously suggested. This allows direct visualization of the RNA without the need for staining and washing of the gel. The buffer system used is MOPS, which unlike Tris-HCl is suitable for use with formaldehyde see Note 1). The RNA is then transferred to a nylon membrane by capillary blotting for subsequent hybridization. Mol-wt standards can be used to estimate transcript size if required.
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References
McMaster, G. K. and Carmicheal, G. G. (1977) Analysis of single-and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74, 4835–4838.
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Fourney, R. M., Miyakoshi, J., Day, R. S., and Paterson, M. C. (1988) Northern blotting: Efficient RNA staining and transfer. Focus 10, 5–7.
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© 1994 Humana Press Inc., Totowa, NJ
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Hodge, R. (1994). Preparation of RNA Gel Blots. In: Isaac, P.G. (eds) Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Methods in Molecular Biology™, vol 28. Humana Press. https://doi.org/10.1385/0-89603-254-X:49
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DOI: https://doi.org/10.1385/0-89603-254-X:49
Publisher Name: Humana Press
Print ISBN: 978-0-89603-254-5
Online ISBN: 978-1-59259-515-0
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