Summary
The inability of structural elements within a reporter mRNA to impede processive decay by the major 5′ and 3′ exonucleases has been a major obstacle to understanding mechanisms of vertebrate mRNA decay. We present here a new approach to this problem focused on quantifying the decay of individual portions of a reporter mRNA. Our approach entails two parts. The first involves the use of a regulated promoter, such as one controlled by tetracycline (tet), to allow reporter gene transcription to be turned off when needed. Cells stably expressing the tet repressor protein are transiently or stably transfected with tet-regulated beta-globin genes in which the sequence element under study is cloned into the 3′-UTR. The second involves the quantification of beta-globin mRNA using the Invader RNA assay, a sensitive and quantitative approach that relies on signal amplification instead of target amplification. Because the Invader RNA assay does not depend on downstream primer binding, the use of multiple probes across the reporter beta-globin mRNA allows for quantification of the decay of individual portions of the mRNA independent of events acting at other sites.
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Acknowledgments
This work was supported by PHS grant R01 GM38277 to D.R.S. E.L.M. was supported in part by PHS grant T32 CA09338, and support for core facilities was provided by PHS center grant P30 CA16058 from the National Cancer Institute to The Ohio State University Comprehensive Cancer Center. We thank Tsetka Takova for help with probe design, troubleshooting, and data analysis, Peggy Eis and David Fritz for help with design of the primary and secondary oligonucleotide probe sets, and Jim Dahlberg and members of the Schoenberg laboratory for helpful discussions. Invader® and Cleavase® are registered trademarks of Third Wave Technologies, Inc.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Murray, E.L., Schoenberg, D.R. (2008). Application of the Invader® RNA Assay to the Polarity of Vertebrate mRNA Decay. In: Wilusz, J. (eds) Post-Transcriptional Gene Regulation. Methods In Molecular Biology™, vol 419. Humana Press. https://doi.org/10.1007/978-1-59745-033-1_18
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DOI: https://doi.org/10.1007/978-1-59745-033-1_18
Publisher Name: Humana Press
Print ISBN: 978-1-58829-783-9
Online ISBN: 978-1-59745-033-1
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