Abstract
The characterization of gene expression patterns within cells and tissues is a very desirable and highly informative way of providing insights into cell development, maintenance, and cell-cell interactions. Several widely used methods, such as Northern or dot blot, in situ hybridization, reverse transcription-polymerase chain reaction (RT- PCR), and ribonuclease protection assay (RPA), have been developed to analyze changing cellular distribution and concentration of mRNAs. The fundamental principle to detect mRNA expression is the specific hybridization of a complementary DNA or RNA probe with its cellular homolog. The relative or absolute quantitation of such mRNAs is complex and each of the techniques has to be judged on its technical limitations and the information it provides. This chapter provides a short introduction to the RPA together with the typical advantages, new nonradioactive approaches, and more simplified gel systems based on original results from our laboratory.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Aranda, M A, Fanle, A, Garcia-Arenal, F, and Malpwa, J in (1995) Experimental evaluation of the rlbonuclease protectlon assay method for the assessment of genetic heterogeneity in populations of RNA viruses Arch Virol 140, 1373–1383
Slsodla, S S, Cleveland, D W, and Sollner-Webb, B (1987) A combmatlon of RNase H and SI nuclease circumvents an artefact inherent to conventional Sl analysis of RNA splicing Nucleic Acids Res 15, 1995–2011
Friedberg, T, Grassow, M A, and Oesch, F (1990) Selective detection of mRNA forms encoding the maJor phenobarbital mduclble cytochrome P450 and other members of the P45OllB family by the RNase A protection assay Arch Biochem Biophys 279, 167–172
Sambrook, J, Fntsch, E F, and Mamatls, T (1989) Molecular Cloning A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Hames, D S and Gillespie, D H (1992) RNA abundance measured by a lysate RNase protectlon assay Biotechniques 12,736–741
Yang, H and Melera, P W (1992) Apphcatlon of the polymerase chain reaction to the ribonuclease protection assay Biotechniques 13, 922–927
Tymms, M J (1995) Quantitative measurement of mRNA using the RNase protection assay, in Methods In Molecular Bzology 37 In Vztro Transcrzptzon and Translatzon Protocols (Tymms, M J, ed ), Humana, Totowa, NJ, pp 32–46
Ma, Y J, Dlssen, G A, Rage F, and OJeda, S R (1996) RNase protection assay Methods A Companzon to Methods in Enzymology 10,273–278
Plath, A, Peters, F, and Emspamer, R (1996) Detection and quantltatlon of specific mRNAs by rlbonuclease protection assay using denaturing horizontal polyacrylamlde gel electrophoresls a radioactive and nonradloactlve approach Electrophoresis 17,471,472
Turnbow, M A and Garner, C W (1993) Ribonuclease protection assay use of blotmylated probes for the detection of two messenger RNAs Biotechniques 15,267–270
Belm, D (1996) The RNase protection assay, in Methods in Molecular Bzology, vol 58 Basic DNA and RNA Protocols (Harwood, A, ed ), Humana, Totowa, NJ, pp 131–136
Pape, M E, Melchlor, G W, and Marottl, K R (1991) mRNA quantltation by a simple and sensitive RNase protection assay Genet Anal Tech Appl 8,206–213
Ludlam, W H, Zang, Z, McCarson, K, Krause, J E, Spray, D C, and Kessler, J A (1994) mRNAs encoding muscarmlc and substance P receptors in cultured sympathlc neurons are dlfferentlally regulated by LIF or CNTF Dev Biol 164,528–539
Melsinger, C and Grothe, C (1996) A sensltlve RNase protectlon assay using 33P labeled antisense riboprobes Mel Biotechnol 5,289–291
Genovese, C, Brufsky, A, Shapn-o, J, and Rowe, D (1989) Detection of mutations in human type I collagen mRNA in osteogenesls Imperfecta by Indirect RNase protection J Biol Chem 264,9632–9631
Wundrack, I and Dooley, S (1992) Nonradloactlve rlbonuclease protection analysis using dlgoxygenm labelling and chemllummescent detection Electrophoresis 13,637,638
Souaze, E, Ntodou-Thome, A, Tran, C Y, Rostene, W, and Forgez, P (1996) Quantitative RT-PCR limits and accuracy Biotechniques 21,280–285
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Humana Press Inc , Totowa, NJ
About this protocol
Cite this protocol
Einspanier, R., Plath, A. (1998). Detecting mRNA by Use of the Ribonuclease Protection Assay (RPA). In: Rapley, R., Walker, J.M. (eds) Molecular Biomethods Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1007/978-1-59259-642-3_5
Download citation
DOI: https://doi.org/10.1007/978-1-59259-642-3_5
Publisher Name: Humana Press
Print ISBN: 978-0-89603-501-0
Online ISBN: 978-1-59259-642-3
eBook Packages: Springer Book Archive