Abstract
Ribonuclease protection assay (RPA) is a sensitive solution hybridization method for quantitation of specific RNAs (1–3). 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.
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References
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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).
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.
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.
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© 1999 Humana Press Inc., Totowa, NJ
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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
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DOI: https://doi.org/10.1385/1-59259-247-3:201
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