Identification of Viroids by Gel Electrophoresis

Robert A. Owens1

1 Molecular Plant Pathology Laboratory (USDA/ARS), Beltsville Agricultural Research Center, Beltsville, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16G.1
DOI:  10.1002/9780471729259.mc16g01s10
Online Posting Date:  August, 2008
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Abstract

Two‐dimensional PAGE involving an initial fractionation under nondenaturing conditions followed by a second electrophoresis under denaturing conditions provides a powerful means to detect viroids and other small circular RNAs. This unit describes a method known as “R(eturn) PAGE” in which denaturation is achieved by simultaneously raising the temperature and lowering the ionic strength during the second electrophoresis. Under denaturing conditions, circular RNAs migrate more slowly than their corresponding linear forms. Following fractionation, RNAs are visualized by staining with ethidium bromide, SYBR Gold, or silver nitrate. Unlike nucleic acid hybridization or RT‐PCR, viroid identification by R‐PAGE requires no nucleotide sequence information. Curr. Protoc. Microbiol. 10:16G.1.1‐16G.1.9. © 2008 by John Wiley & Sons, Inc.

Keywords: viroids; polyacrylamide gel electrophoresis (PAGE); circular RNAs; (R)eturn PAGE

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: RNA Isolation
  • Basic Protocol 2: R‐PAGE Analysis
  • Basic Protocol 3: Visualize Small Circular RNAs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: RNA Isolation

  Materials
  • White quartz sand (50 to 70 mesh; Sigma, cat. no. 14808‐60‐7 or equivalent)
  • Liquid nitrogen
  • Plant tissue (fresh or frozen)
  • TRIzol reagent (Invitrogen)
  • Chloroform (molecular‐biology‐grade)
  • Isopropanol (molecular‐biology‐grade)
  • 70% (v/v) ethanol/0.1 M sodium acetate (see recipe)
  • RNase‐free H 2O
  • 4 M LiCl
  • Absolute ethanol
  • Mortar and pestle (autoclaved)
  • Spectrophotometer
  • Additional reagents and equipment for measuring A 260 using a spectrophotometer (Gallagher and Desjardins, )

Basic Protocol 2: R‐PAGE Analysis

  Materials
  • GlassPlus or equivalent glass cleaner
  • 95% ethanol
  • Gel solution (see Table 16.1.1)
  • 1× TBE
  • Isolated RNA ( protocol 1)
  • 6× gel loading buffer (see recipe)
  • 0.5 µg/ml ethidium bromide in water
  • Compatible plates, spacers, and combs designed for Bio‐Rad's Mini PROTEAN 3 apparatus (available from Bio‐Rad Laboratories)
  • JGC‐3 Joey Gel Casting System (Owl Separation Systems), optional
  • P8DS Emperor Penguin Vertical Electrophoresis System (Owl Separation Systems)
  • Micropipet equipped with a drawn‐out plastic tips
  • Haake Model DC10‐P5/U circulating baths (available from Fisher Scientific) or any circulating bath capable of maintaining temperatures between 25° and 100°C is also acceptable
    Table 6.0.1   MaterialsVolumes of Reagents Used to Cast Polyacrylamide Gels for R‐PAGE

    Component 5% 7.5% 10%
    40% (w/v) acrylamide stock (see recipe) 3.1 ml 4.7 ml 6.25 ml
    10× TBE buffer (see recipe) 2.5 ml 2.5 ml 2.5 ml
    H 2O 19.3 ml 17.7 ml 16.1 ml
    TEMED aa 12.5 µl 12.5 µl 12.5 µl
    10% (w/v) ammonium persulfate (APS) bb 125 µl 125 µl 125 µl
    Total 25 ml 25 ml 25 ml

     aN,N, N′,N′‐Tetramethylethylenediamine.
     bAPS should be added last.

Basic Protocol 3: Visualize Small Circular RNAs

  Materials
  • Electrophoresed gel ( protocol 2)
  • Staining solution (0.5 µg/ml ethidium bromide in water)
  • Scalpel or single‐edged razor blade
  • Plastic or glass dish for staining
  • Platform shaker
  • Plastic wrap
  • UV transilluminator
  • Additional reagents and equipment for photographing the gel (Voytas, )
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Figures

Videos

Literature Cited

   Brown, T., Mackey, K., and Du, T. 2004. Analysis of RNA by northern and slot blot hybridization. Curr. Protoc. Mol. Biol. 67:4.9.1‐4.9.19.
   Feldstein, P.A., Levy, L., Randles, J.W., and Owens, R.A. 1997. Synthesis and two‐dimensional electrophoretic analysis of mixed populations of circular and linear RNAs. Nucleic Acids Res. 25:4850‐4854.
   Gallagher, S.R. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1‐10.2A.37.
   Gallagher, S.R and Dejardins, P.R. 2006. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr. Protoc. Mol. Biol. 76:A.3D.1‐A.3D.21.
   Hanold, D., Semancik, J.S., and Owens, R.A. 2003. Polyacrylamide gel electrophoresis. In Viroids (A. Hadidi, R. Flores, J.W. Randles, and J.S. Semancik, eds.) pp. 95‐102. CSIRO Publishing, Collingwood, Australia.
   Kingston, R.E., Chomczynski, P., and Sacchi, N. 1996. Guanidine methods for total RNA preparation. Curr. Protoc. Mol. Biol. 36:4.2.1‐4.2.9.
   Navarro, B. and Flores, R. 1997. Chrysanthemum chlorotic mottle viroid: Unusual structural properties of a subgroup of self‐cleaving viroids with hammerhead ribozymes. Proc. Natl. Acad. Sci. U.S.A. 94:11622‐11267.
   Rivera‐Bustamante, R., Gin, R., and Semancik, J.S. 1986. Enhanced resolution of circular and linear molecular forms of viroid and viroid‐like RNA by electrophoresis in a discontinuous‐pH system. Anal. Biochem. 156:91‐95.
   Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual (Third edition), pp 12.74‐12.80. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Schmitz, A. and Riesner, D. 2006. Purification of nucleic acids by selective precipitation with polyethylene glycol 6000. Anal Biochem. 354:311‐313.
   Schumacher, J., Meyer, N., Riesner, D., and Weidemann, H.L. 1986. Diagnostic procedure for detection of viroids and viruses with circular RNAs by ‘return’‐gel electrophoresis. J. Phytopathol. 115:332‐343.
   Singh, R.P. and Boucher, A. 1987. Electrophoretic separation of a severe from mild strains of potato spindle tuber viroid. Phytopathology 77:1588‐1591.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
Key References
   Flores, R. and Owens, R.A. 2008. Viroids. In Encyclopedia of Virology, Third Edition (B.W.J. Mahy and M.H.V. Van Regenmortel, eds.). Elsevier/Academic Press, London, in press.
  The largest single reference source of current virological knowledge for both specialist and general readers.
   Hadidi, A., Flores, R., Randles, J.W., and Semancik, J.S. (eds.) 2003. Viroids. CSIRO Publishing, Collingwood, Australia.
  This monograph provides a comprehensive overview of the biological and molecular properties of viroids. Most complete single source of information about viroids.
   Steger, G. and Riesner, D. 2003. Molecular characteristics. In Viroids (A. Hadidi, R. Flores, J.W. Randles, and J.S. Semancik, eds.) pp. 15‐29. CSIRO Publishing, Collingwood, Australia.
  A concise summary of the structural properties of viroids.
   Singh, R.P. 2007. Molecular detection of plant viroids. In Biotechnology and Plant Disease Management (Z.K. Punja, S.H. De Boer, and H. Sanfaçon, eds.), pp. 277‐300. CAB International, Wallingford, U.K.
  R‐PAGE and other electrophoretic methods are compared with other detection techniques such as molecular hybridization and RT‐PCR.
Internet Resources
   http://subviral.med.uottawa.ca/
  Subviral RNA Database. Maintained by Martin Pelchat [RNA Group/Groupe ARN, Département de biochimie, Université de Sherbrooke, Sherbrooke (Québec) J1H 5N4, Canada], this website contains the sequences of more than 2400 viroids and related RNAs.
   http://www.ictvonline.org
  The Universal Database of the International Committee on the Taxonomy of Viruses is a convenient source of current information on viroid (and virus) taxonomy.
   http://www.probes.invitrogen.com/handbook/
  Invitrogen Detection Technologies: Contains an informative discussion of nucleic acid staining using SYBR Gold, SYBR Green I, and SYBR Green II.
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