Abstract
When characterizing the developmental expression of a novel gene, or when examining the response of a known gene to experimental manipulations, it is important to be able to assay mRNA transcript levels accurately. Although a number of techniques for transcript analysis are available, one of the most useful and widespread is the ribonuclease (RNase) protection assay (for example, see Fig. 1). The major advantages of RNase protection analysis are good sensitivity, excellent specificity, and the linear response to differing transcript levels. Perhaps the major disadvantage of RNase protection is the need to prepare special RNA probes and the fact that the RNA samples used for RNase protection analysis are destroyed and therefore cannot be reused. In addition to expression analysis, RNase protection can also be applied to a number of additional experimental goals, including mapping of transcription start sites, mapping of intron/exon boundaries, analysis of alternative splicing, and determination of the rate of degradation of nucleic acids introduced into the embryo.
Developmental profile of the EF-1α gene transcript as seen by a 70-min exposure of the final acrylamide gel. The input probe is denoted by an asterisk; the darkened circle marks the level of the protected fragment. Expression can first be detected at stage 10. Note that EF1-α is an exceptionally abundant transcript and most gene products will require a significantly longer exposure time to visualize the protected fragments.
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© 1999 Humana Press Inc.
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Newman, C.S., Krieg, P.A. (1999). Ribonuclease Protection Analysis of Gene Expression in Xenopus . In: Guille, M. (eds) Molecular Methods in Developmental Biology. Methods in Molecular Biology™, vol 127. Humana Press. https://doi.org/10.1385/1-59259-678-9:29
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DOI: https://doi.org/10.1385/1-59259-678-9:29
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