Abstract
Eukaryotic messenger RNA (mRNA) can be separated from the other RNA species in a total RNA preparation by affinity chromatography by virtue of the presence of a polyadenylic acid “tail,” 20–25 bases long, at the 3′ end of the molecule (1). Oligo(dT)-cellulose is used routinely for homemade affinity columns, but a range of ready-made products is now available from a number of suppliers (e.g., Hybond-mAp, Amersham, Arlington Heights, IL; PolyATract™, Promega, Madison, WI). Oligo(dT)-cellulose consists of a polymer of 10–20 T-residues, covalently linked to a cellulose matrix, which will hybridize to and bind poly(A)+ containing RNA providing the tail is at least 15–20 bases long. Ribosomal and transfer RNA do not bind efficiently to oligo(dT) and can be washed through the column. Bound poly(A)+ RNA can then be eluted with low salt buffers. The protocol given below is a general purpose method designed to give high yields of cDNA synthesis-/in vitro translation-quality mRNA from relatively large amounts of starting material (1–2 mg total RNA).
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References
Aviv, H. and Leder, P. (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69, 1408–1412.
Mozer, T. J. (1980) Partial purification and characterisation of the mRNA for a α-amylase from barley aleurone layers. Plant Physiol. 65, 834–837.
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© 1995 Humana Press Inc., Totowa, NJ
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Scott, R.J. (1995). Poly(A)+RNA Isolation. In: Jones, H. (eds) Plant Gene Transfer and Expression Protocols. Methods in Molecular Biology™, vol 49. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-321-X:203
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DOI: https://doi.org/10.1385/0-89603-321-X:203
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-321-4
Online ISBN: 978-1-59259-536-5
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