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Gene Expression Analyzed by Ribonuclease Protection Assay

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Developmental Biology Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 137))

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Abstract

The ribonuclease (RNase) protection assay provides a highly sensitive method for the detection and quantitation of specific RNAs from tissues and cells as well as for the analysis of mRNA and gene structure (1). This solution hybridization approach is at least 10-fold more sensitive than Northern blot analysis, and thus is useful for the evaluation of low-abundance mRNAs. Furthermore, the greater stability of the RNA duplex structure over RNA-DNA hybrids used in S1 nuclease protection assays provides the greatest sensitivity among the solution hybridization approaches. Structurally, these assays also provide a useful means of determining the size of specific exons (2,3) in a gene and for the quantitative analysis of specific alternative splicing events occurring in homogeneous tissues (see Note 1), as well as the mapping of transcription start site (4,5).

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References

  1. Zinn K., DiMaio D., and Maniatis T. (1983) Identification of two distinct regulatory regions adjacent to the human b-interferon gene. Cell 34, 865–879.

    Article  PubMed  CAS  Google Scholar 

  2. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., and Green M. R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 12, 7035–7056.

    Article  PubMed  CAS  Google Scholar 

  3. Lau E. T., Kong R. Y. C., and Cheah K. S. E. (1993) Acritical assessment of the RNase protection assay as a means of determining exon sizes. Anal. Biochem. 209, 360–366.

    Article  PubMed  CAS  Google Scholar 

  4. Bennett V. D., Pallante K. M., and Adams S. L. (1991) The splicing pattern of fibronectin mRNA changes during chondrogenesis resulting in an unusual form of the mRNA in cartilage. J. Biol. Chem. 266, 5918–5924.

    PubMed  CAS  Google Scholar 

  5. Gehris A. L., Brandli D. W., Lewis S. D., and Bennett V. D. (1996) The exon encoding the fibronectin type III-9 repeat is constitutively included in the mRNA from chick limb mesenchyme and cartilage. Biochim. Biophys. Acta 1311, 5–12.

    Article  PubMed  Google Scholar 

  6. Norton P. A. and Hynes R. O. (1987) Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells. Mol. Cell. Biol. 7, 4297–4307.

    PubMed  CAS  Google Scholar 

  7. Gilman M. (1993) Gel purification of RNA probes, in Current Protocols in Molecular Biology, Wiley New York, pp.4.7.3–4.7.4.

    Google Scholar 

  8. Davis M. J. J., Bailey C. S., and Smith C. K. (1997) Use of internal controls to increase quantitative capabilities of the ribonuclease protection assay. Biotechniques 23, 280–285.

    PubMed  CAS  Google Scholar 

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© 2000 Humana Press Inc., Totowa, NJ

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Bennett, V.D. (2000). Gene Expression Analyzed by Ribonuclease Protection Assay. In: Tuan, R.S., Lo, C.W. (eds) Developmental Biology Protocols. Methods in Molecular Biology™, vol 137. Humana Press. https://doi.org/10.1385/1-59259-066-7:45

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  • DOI: https://doi.org/10.1385/1-59259-066-7:45

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-854-7

  • Online ISBN: 978-1-59259-066-7

  • eBook Packages: Springer Protocols

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