Abstract
Whereas Northern analysis is a standard procedure by which abundant levels of mRNA can be quantified and characterized by size, there is a limit to the sensitivity of this technique, even with the best probes and the use of poly(A+) mRNA enrichment (see Chapters 28 and 30). At these times, it is necessary to move to more sensitive techniques. RNase protection assays or equivalent are much more sensitive but are technically difficult to perform, and require expensive dedicated equipment as well as production of riboprobes. More recently, there has been a predominance of the use of equally sensitive reverse transcription-polymerase chain reaction (RT-PCR) methodologies, which rely on the now more universally available PCR cycler and otherwise inexpensive and easily performed Southern Blot analytical methodology.
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
Tsai, S. J. and Wiltbank, M. C. (1996) Quantification of mRNA using competitive RT-PCR with standard curve methodology. Biotechniques 21, 862–866.
Mallet, F., Oriol, G., Mary, C., Verrier, B., and Mandrand, B. (1995) Continuous RT-PCR using AMV-RT and Taq DNA polymerase: characterization and comparison to uncoupled procedures. Biotechniques 18, 678–687.
Bird, I. M., Zheng, J., Cale, J. M., and Magness, R. R. (1997) Pregnancy induces an increase in angiotensin II type-1 receptor expression in uterine but not systemic artery endothelium. Endocrinology 138, 490–498.
Zheng, J., Bird, I. M., Melsaether, A. N., and Magness, R. R. (1998) Activation of the mitogen-acitivated protein kinase cascade is nescessary but not sufficient for bFGF and EGF stimulated expression of endothelial nitric oxide synthase. J. Biol. Chem., submitted.
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
Cale, J.M., Shaw, C.E., Bird, I.M. (1998). Optimization of a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Mass Assay for Low-Abundance mRNA. In: Bird, I.M. (eds) Phospholipid Signaling Protocols. Methods in Molecular Biology™, vol 105. Humana Press. https://doi.org/10.1385/0-89603-491-7:351
Download citation
DOI: https://doi.org/10.1385/0-89603-491-7:351
Publisher Name: Humana Press
Print ISBN: 978-0-89603-491-4
Online ISBN: 978-1-59259-255-5
eBook Packages: Springer Protocols