Abstract
For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. In vitro transcription or chemical synthesis are the principal methods to produce RNA. Here, we describe an alternative method allowing RNA production in bacteria and its purification by liquid chromatography. In a few days, between 10 and 100 mg of pure RNA are obtained with this technique.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Schenborn ET, Mierendorf RC Jr (1985) A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res 16:6223–6236
Lukavsky PJ, Puglisi JD (2004) Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10:889–893
Cheong HK, Hwang E, Lee C, Choi BS, Cheong C (2004) Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography. Nucleic Acids Res 32:e84
Kieft JS, Batey RT (2004) A general method for rapid and nondenaturing purification of RNAs. RNA 10:988–995
Masson J-M, Miller JH (1986) Expression of synthetic tRNA genes under the control of a synthetic promoter. Gene 47:179–183
Meinnel T, Mechulam Y, Fayat G (1988) Fast purification of a functional elongator tRNAmet expressed from a synthetic gene in vivo. Nucleic Acids Res 16:8095–8096
Tisné C, Rigourd M, Marquet R, Ehresmann C, Dardel F (2000) NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription. RNA 6:1403–1412
Wallis NG, Dardel F, Blanquet S (1995) Heteronuclear NMR studies of the interactions of 15N-labeled methionine-specific transfer RNAs with methionyltRNA transformylase. Biochemistry 34:7668–7677
Engelke DR, Hopper AK (2006) Modified view of tRNA: stability amid sequence diversity. Mol Cell 21:144–145
Ponchon L, Dardel F (2007) Recombinant RNA technology: the tRNA scaffold. Nat Methods 4:571–576
McKenna SA, Kim I, Puglisi EV, Lindhout DA, Aitken CE, Marshall RA, Puglisi JD (2007) Purification and characterization of transcribed RNAs using gel filtration chromatography. Nat Protoc 2:3270–3277
Ponchon L, Beauvais G, Nonin-Lecomte S, Dardel F (2009) A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold. Nat Protoc 4:947–959
Neidhardt FC, Fraenkel D (1961) Metabolic regulation of RNA synthesis in bacteria. Cold Spring Harb Symp Quant Biol 26:63–74
Tisné C, Rigourd M, Marquet R, Ehresmann C, Dardel F (2000) NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription. RNA 6:1403–1412
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ponchon, L., Dardel, F. (2013). Purification of RNA Expressed In Vivo Inserted in a tRNA Scaffold. In: Conn, G. (eds) Recombinant and In Vitro RNA Synthesis. Methods in Molecular Biology, vol 941. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-113-4_1
Download citation
DOI: https://doi.org/10.1007/978-1-62703-113-4_1
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-112-7
Online ISBN: 978-1-62703-113-4
eBook Packages: Springer Protocols