Skip to main content

RNase Protection Assays for Quantitation of Gene Expression in Vascular Tissue

  • Protocol
Vascular Disease

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 30))

  • 484 Accesses

Abstract

Ribonuclease protection assay (RPA) is a sensitive solution hybridization method for quantitation of specific RNAs (13). The method is based on the ability of single-strand specific ribonuclease to degrade single-stranded RNA while leaving intact fragments of labeled antisense RNA probe, which are annealed to homologous sequence in the sample RNA. After ribonuclease digestion, the hybridized portion of the probe (“protected fragment”) can be visualized via electrophoresis of the mixture on a denaturing polyacrylamide gel followed by autoradiography.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 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. Nucleic Acids Res. 12, 7035–7056.

    Article  CAS  PubMed  Google Scholar 

  2. Sambrook, J., Fritsch, E. F.m and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  3. Gilman, M. (1989) In: Current Protocols in Molecular Biology, (Ausubel, F., et al., eds.), Vol. 2, Wiley, New York, NY, pp. 4.7.1–4.7.8.

    Google Scholar 

  4. Leech, C. J. and Faber, J. E. (1996) Different α-adrenoceptor subtypes mediate constriction of arterioles and venules. Am. J. Physiol. 270 (Heart Circ. Physiol. 39), H710–H722.

    CAS  PubMed  Google Scholar 

  5. Rhodin, J. A. G. (1980) Architecture of the vessel wall. In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. American Physiological Society, Bethesda, MD, pp. 1–32.

    Google Scholar 

  6. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Ann. Biochem. Exp. Med. 162, 156–159.

    Article  CAS  Google Scholar 

  7. Chen, L., Xin, X., Eckhart, A. D., Yang, N., and Faber, J. E. (1995) Regulation of vascular smooth muscle growth by α1-adrenoceptor subtypes in vitro and in situ. J. Biol. Chem. 270, 30,980–30,985.

    Article  CAS  PubMed  Google Scholar 

  8. Xin, X., Yang, N., Eckhart, A. D., and Faber, J. E. (1997) α1D-Adrenergic receptors and mitogen-activated protein kinase mediate increased protein synthesis by arterial smooth muscle. Mol. Pharmacol. 51, 764–775.

    CAS  PubMed  Google Scholar 

  9. Yang, N., Xin, X., Eckhart, A. D., and Faber, J. E. (1997) CRE-like response element regulates expression of rat α2D-adrenergic receptor gene in vascular smooth muscle. Am. J. Physiol. 273 (Heart Circ. Physiol 42), H85–H95.

    CAS  PubMed  Google Scholar 

  10. Faber, J. E., Yang, N., and Erami, C. (1999) Angioplasty injury reduces α-adrenergic receptor (AR) expression in neointima, media and adventitia. FASEB J 13, 416.12 (abstract).

    Google Scholar 

  11. Piascik, M. T., Guarino, R. D., Smith, M. S., Soltis E. E., Saussy, D. L. Jr, and Perez, D. M. (1995) The specific contribution of the novel alpha-1D adrenoceptor to the contraction of vascular smooth muscle. J. Pharm. Exper. Thera. 275, 1583–1589.

    CAS  Google Scholar 

  12. Yang, N., Erami, C., and Faber, J. E. (1999) Adventitial fibroblasts (AFBs) express functional α1-adrenergic receptors (ARs), and modulate to myofibroblasts (myoFBs) in primary culture and after angioplasty in vivo. FASEB J. 13, 426.13 (abstract).

    Google Scholar 

  13. Stewart, A. F., Rokosh, D. G., Bailey, B. A., Karns, L. R., Chang, K. C., Long, C. S., et al. (1994) Cloning of the rat alpha 1C-adrenergic receptor from cardiac myocytes. alpha 1C, alpha 1B, and alpha 1D mRNAs are present in cardiac myocytes but not in cardiac fibroblasts. Circ. Res. 75(4), 796–802.

    CAS  PubMed  Google Scholar 

  14. Clements, M. L., Banes, A. J., and Faber, J. E. (1997) Effects of mechanical loading on vascular α1D-and α1D-adrenergic receptor expression. Hypertension 29, 1156–1164.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Yang, N., Wang, S., Faber, J.E. (1999). RNase Protection Assays for Quantitation of Gene Expression in Vascular Tissue. In: Baker, A.H. (eds) Vascular Disease. Methods in Molecular Medicine™, vol 30. Humana Press. https://doi.org/10.1385/1-59259-247-3:201

Download citation

  • DOI: https://doi.org/10.1385/1-59259-247-3:201

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-731-1

  • Online ISBN: 978-1-59259-247-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics