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RNA pp 293–309Cite as

Polysome Analysis and RNA Purification from Sucrose Gradients

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 703))

Abstract

Velocity separation of translation complexes in linear sucrose gradients is the ultimate method for both analysis of the overall fitness of protein synthesis as well as for detailed investigation of physiological roles played by individual factors of the translational machinery. Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and Western blotting, genome-wide microarray analysis or qRT-PCR. This chapter provides a basic overview of the polysome profile analysis technique and the RNA isolation procedure from sucrose gradients. We also discuss possible experimental pitfalls of data normalization, describe main alternatives of the basic protocol and outline a novel application of denaturing RNA electrophoresis in several steps of polysome profile analysis.

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Acknowledgment

This work was supported by Czech Science Foundation grant No. 301/07/0607, Ministry of Education, Youth and Sports of the Czech Republic grant No. LC06066 (both to MP). LV was supported by The Wellcome Trusts grant No. 076456/Z/05/Z, Fellowship of Jan E. Purkyne from Academy of Sciences of the Czech Republic, and Inst. Research Concept AV0Z50200510.

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Authors and Affiliations

  1. Department of Genetics and Microbiology, Charles University in Prague, Prague, Czech Republic

    Tomáš Mašek

  2. Laboratory of Regulation of Gene Expression, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Leoš Valášek

  3. Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic

    Martin Pospíšek

Authors
  1. Tomáš Mašek
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  2. Leoš Valášek
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  3. Martin Pospíšek
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Corresponding author

Correspondence to Tomáš Mašek .

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Editors and Affiliations

  1. The Panum Institute, Dept. of Cellular & Molecular Medicine, University of Copenhagen, Blegdamsvej 3, Copenhagen, 2200N, Denmark

    Henrik Nielsen

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Mašek, T., Valášek, L., Pospíšek, M. (2011). Polysome Analysis and RNA Purification from Sucrose Gradients. In: Nielsen, H. (eds) RNA. Methods in Molecular Biology, vol 703. Humana Press. https://doi.org/10.1007/978-1-59745-248-9_20

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  • DOI: https://doi.org/10.1007/978-1-59745-248-9_20

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-913-0

  • Online ISBN: 978-1-59745-248-9

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