Extracting Viral RNAs from Plant Protoplasts

Marc R. Fabian1, K. Andrew White1

1 York University, Toronto, Canada
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16E.1
DOI:  10.1002/9780471729259.mc16e01s6
Online Posting Date:  August, 2007
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Abstract

The analysis of viral RNA is a fundamental aspect of plant RNA virus research. Studies that focus on viral RNAs often involve virus infections of plant protoplasts (see UNITS 16D.1‐16D.4). Protoplast offer the advantage of simultaneous initiation of infections, which allows for superior temporal and quantitative analyses of viral RNAs. The efficient isolation of intact viral RNA is key to any such investigations. This unit describes two basic protocols for extracting viral RNAs from plant protoplasts. An approach for preparing double‐stranded viral RNA from total RNA pools is also provided. The viral RNA prepared by using these techniques can be used for further analyses such as primer extension, reverse transcription–PCR, and northern blotting. Curr. Protoc. Microbiol. 6:16E.1.1‐16E.1.6. © 2007 by John Wiley & Sons, Inc.

Keywords: RNA; protoplast; isolation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Total Nucleic Acids Using EDTA‐Detergent Lysis and Phenol/Chloroform Extraction
  • Basic Protocol 2: Isolation of Total RNA Using Guanidinium Isothiocyanate Solution
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Isolation of Total Nucleic Acids Using EDTA‐Detergent Lysis and Phenol/Chloroform Extraction

  Materials
  • Infected protoplasts (see units 16.116.D.1 16.416.D.4)
  • Nucleic acid extraction buffer (see recipe)
  • 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol (PCI; appendix 2A)
  • 1 M and 3M sodium acetate, pH 5.2
  • 95% and 70% ethanol, ice‐cold
  • Diethylpyrocarbonate (DEPC)‐treated H 2O ( appendix 2A)
  • 7 M lithium chloride (LiCl)
  • 2× DNase buffer (see recipe)
  • RNase‐free DNase I (10 U/µl; bovine pancreas, Amersham Biosciences)
  • 1.5‐ml microcentrifuge tubes
  • Standard microcentrifuge
  • Tube vortexer
  • Additional reagents and equipment for performing agarose gel electrophoresis and
  • ethidium‐bromide staining (Voytas, ) and northern blot analysis (Brown et al., )
NOTE: All reagents and vessels used should be sterile and RNase‐free.

Basic Protocol 2: Isolation of Total RNA Using Guanidinium Isothiocyanate Solution

  Materials
  • Infected protoplasts (see units 16.116.D.1 16.416.D.4)
  • Guanidinium solution (see recipe)
  • 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol (PCI; appendix 2A)
  • 2 M sodium acetate, pH 4 ( appendix 2A)
  • DEPC‐treated water ( appendix 2A)
  • Isopropanol, ice‐cold
  • 70% ethanol, ice‐cold
  • 1.5‐ml microcentrifuge tubes
  • Tube vortexer
  • Additional reagents and equipment for performing agarose gel electrophoresis and
  • ethidium‐bromide staining (Voytas, ) and northern blot analysis (Brown et al., )
NOTE: Use sterile, RNase‐free reagents and vessels.
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Figures

Videos

Literature Cited

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.
   Chomczynski, P. and Sacchi, N. 1987. Single‐Step method of RNA isolation by acid guanidinium thiocyanate‐ phenol‐chloroform extraction. Anal. Biochem. 162:156‐159.
   Morris, T.J. and Dodds, J.A. 1979. Isolation and analysis of double‐stranded RNA from virus‐infected plant and fungal tissue. Phytopathology 69:854‐858.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
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