Northern Analysis of Viral Plus‐ and Minus‐Strand RNAs

Paramasivan Vijaya Palani1, Na‐Sheng Lin1

1 Institute of Plant and Microbial Biology, Taipei
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16E.3
DOI:  10.1002/9780471729259.mc16e03s4
Online Posting Date:  February, 2007
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Abstract

Replication is a fundamental activity of viruses. Replication of positive‐sense RNA viruses involves the synthesis of complementary minus‐strand intermediates from the parental RNA template followed by synthesis of nascent plus strands. Negative‐sense RNA genome and double‐stranded RNA are copied into positive‐sense mRNA before translation. To detect and estimate the abundance of plus‐ and minus‐strand viral transcripts in the infected samples, northern analysis is the most commonly used method.

Keywords: plus‐/minus‐strand RNA detection; glyoxalated RNA; agarose‐formaldehyde gel electrophoresis; northern blotting

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Northern Analysis of Plus‐ and Minus‐Strand
  • Support Protocol 1: Extraction of RNA from Plant Tissue
  • Support Protocol 2: Separation of Poly(A) RNA from Total RNA
  • Support Protocol 3: Northern Hybridization of Glyoxalated RNA Fractionated on a Nondenaturing Agarose Gel
  • Support Protocol 4: Northern Hybridization of RNA Fractionated on an Agarose‐Formaldehyde Gel
  • Support Protocol 5: Northern Hybridization of Formamide‐Denatured RNA Fractionated on a Nondenaturing Agarose Gel
  • Support Protocol 6: RNA Transfer Using an Upward Capillary System
  • Support Protocol 7: RNA Transfer Using a Downward Capillary System
  • Support Protocol 8: Preparation of Oligonucleotide Probe by End‐Labeling
  • Support Protocol 9: Preparation of Riboprobe by In Vitro Transcription
  • Support Protocol 10: Preparation of Total RNA to Detect Low Abundant RNA Species
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Northern Analysis of Plus‐ and Minus‐Strand

  Materials
  • Sample RNA ( protocol 2 or protocol 32)
  • Methylene blue solution (see recipe)
  • 0.2× SSC ( appendix 2A)
  • 1% (w/v) SDS
  • Prehybridization solution (see recipe)
  • Low‐stringency wash buffer (see recipe)
  • High‐stringency wash buffer (see recipe)
  • Strip buffer (see recipe), 70° to 75°C
  • Blotting paper
  • UV crosslinker (Stratagene), optional
  • 80°C vacuum oven, optional
  • Microwave oven, optional
  • Glass tray
  • Camera with yellow filter, optional
  • Hybridization tube
  • 65°C hybridization oven with rotation
  • Plastic wrap
  • X‐ray film (e.g., Kodak Biomax MS film) and cassette (e.g., Hypercasette, Amersham Biosciences), optional
  • Densitometer (e.g., GS‐700, BioRad), optional
  • PhosphorImager (Molecular Dynamics, Amersham Pharmacia Biotech)
  • Additional reagents and equipment for electrophoresis (see Support Protocols protocol 43 to protocol 65); membrane transfer (see Support Protocols protocol 76 and protocol 87); DNA/RNA probe preparation (see Support Protocols protocol 98 and protocol 109)

Support Protocol 1: Extraction of RNA from Plant Tissue

  Materials
  • Infected plant tissues
  • Liquid nitrogen
  • RNA extraction buffer (see recipe), 80°C
  • 24:1 (v/v) chloroform/isoamyl alcohol mixture, 4°C
  • 4 M lithium chloride (LiCl)
  • DEPC‐treated water ( appendix 2A)
  • 3 M sodium acetate, pH 5.2
  • Isopropanol/100% ethanol, ice cold
  • 70% ethanol
  • 10 U/µl RNase‐free DNase I (Roche)
  • 0.1% to 0.5% SDS in TE buffer, pH 7.6 or 0.1 mM EDTA, pH 7.5
  • Deionized formamide
  • Sterile mortar and pestle
  • 15‐ml propylene snap‐cap tube
  • Centrifuge
  • Micropipettor

Support Protocol 2: Separation of Poly(A) RNA from Total RNA

  Materials
  • Oligo(dT) cellulose (Ambion, Invitrogen)
  • 10 N sodium hydroxide (NaOH)
  • 40 U/µl RNasin (Promega)
  • DEPC‐treated water ( appendix 2A)
  • 1× column loading buffer (see recipe)
  • Total RNA ( protocol 2)
  • Elution buffer (see recipe)
  • 100% ethanol
  • Platform rocker or rotator
  • Disposable poly‐prep columns (Bio‐Rad)

Support Protocol 3: Northern Hybridization of Glyoxalated RNA Fractionated on a Nondenaturing Agarose Gel

  Materials
  • Agarose (SeaKem)
  • 1× sodium phosphate buffer, pH 6.3
  • Sample RNA, salt‐free ( protocol 2 or protocol 32)
  • DEPC‐treated water ( appendix 2A)
  • Standards
  • Markers
  • Glyoxal reaction mix (see recipe)
  • Nondenaturing gel loading buffer (see recipe)
  • 10× SSC ( appendix 2A)
  • 55°C water bath
  • 2‐mm comb
  • Horizontal electrophoresis apparatus for 20 × 10–cm gels (built‐in recirculation system, optional)
  • Peristaltic pump, optional
  • 1.5‐ml microcentrifuge tubes
  • Glass plates

Support Protocol 4: Northern Hybridization of RNA Fractionated on an Agarose‐Formaldehyde Gel

  Materials
  • Agarose (SeaKem)
  • DEPC‐treated water ( appendix 2A)
  • 1× and 10× MOPS buffer, pH 8.0 (see recipe)
  • Formladehyde (deionized; see recipe)
  • Sample RNA, salt‐free ( protocol 2 or protocol 32)
  • Standards
  • Markers
  • Formamide, stored indefinitely at –20°C
  • Formaldehyde gel loading buffer (see recipe)
  • 55°C water bath
  • Horizontal electrophoresis apparatus with 20 × 10–cm gel capacity (built‐in recirculation system, optional)
  • 2‐mm comb
  • Peristaltic pump, optional
  • 1.5‐ml microcentrifuge tubes
  • Glass plate

Support Protocol 5: Northern Hybridization of Formamide‐Denatured RNA Fractionated on a Nondenaturing Agarose Gel

  Materials
  • Agarose (SeaKem)
  • 1× sodium phosphate buffer, pH 6.3 ( appendix 2A)
  • Sample RNA, salt‐free ( protocol 2 or protocol 32)
  • DEPC‐treated water ( appendix 2A)
  • 50% to 70% formamide, stored indefinitely at –20°C
  • Standards
  • Markers
  • Nondenaturing gel loading buffer (see recipe)
  • 6% formladehyde (deionized; see recipe)
  • 10× SSC ( appendix 2A)
  • 55°C water bath
  • Horizontal electrophoresis apparatus with 20 × 10–cm gel capacity (built‐in recirculation system, optional)
  • 2‐mm comb
  • Peristaltic pump, optional
  • 1.5‐ml microcentrifuge tubes
  • Glass plate

Support Protocol 6: RNA Transfer Using an Upward Capillary System

  Materials
  • 2× and 20× SSC ( appendix 2A)
  • Gel (Support Protocols protocol 43 to protocol 65)
  • Nylon membrane
  • Glass plates
  • Plastic wrap
  • Blotting paper (Schleicher & Schuell GB002)
  • Thick blotting paper (Whatman 3 MM, Schleicher & Schuell GB004)
  • Glass rod or pipet
  • Paper towels and ∼500‐g weight

Support Protocol 7: RNA Transfer Using a Downward Capillary System

  Materials
  • Nylon membrane
  • Gel (see Support Protocols protocol 43 to protocol 65)
  • 20× SSC ( appendix 2A)
  • Glass tray (30 × 20–cm)
  • Thick blotting paper (Whatman 3 MM, Schleicher & Schuell GB004)
  • Blotting paper (Schleicher & Schuell GB002)
  • Glass rod or pipet
  • Resevoir troughs
  • Thin glass plate

Support Protocol 8: Preparation of Oligonucleotide Probe by End‐Labeling

  Materials
  • 100 pmol/µl synthetic oligonucleotides
  • Bacteriophage T4 polynucleotide kinase and 10× bacteriophage T4 polynucleotide kinase buffer (New England BioLabs)
  • >5000 Ci/mmol [γ‐32P]ATP
  • 1 M Tris·Cl, pH 8.0 ( appendix 2A)
  • 5 M ammonium acetate
  • 70% and 100% ethanol, ice cold
  • TE buffer, pH 7.6 ( appendix 2A)
  • 1.5‐ml microcentrifuge tubes
  • 37° and 68°C water baths
  • Additional reagents and equipment to measure specific activity (see protocol 10)

Support Protocol 9: Preparation of Riboprobe by In Vitro Transcription

  Materials
  • Plasmid DNA
  • Restriction enzymes
  • DEPC‐treated water ( appendix 2A)
  • 5 mM ATP, GTP, and UTP ( appendix 2A)
  • 100 µM CTP
  • 10× transcription buffer
  • 20 mCi/µl or 3000 Ci/mM [α‐32P] rCTP
  • 40 U/µl RNasin (Promega)
  • 15 U/µl T7/SP6 DNA‐dependent RNA polymerase
  • 10 U/µl RNase‐free DNase I (Roche)
  • Scintillation fluid
  • 37°C (for T7/T3 polymerase) or 40°C (for SP6 polymerase) water bath
  • Nuc‐Trap push column (Stratagene), optional
  • Scintillation counter
  • Whatman filter paper disk (DE81 filter, Whatman)
  • Additional reagents and equipment for restriction digestion and purifying DNA (Moore and Dowhan, )

Support Protocol 10: Preparation of Total RNA to Detect Low Abundant RNA Species

  Materials
  • Sample RNA ( protocol 2)
  • 32P‐labeled riboprobe (see protocol 10)
  • Annealing buffer A (see recipe)
  • RNase A
  • Annealing buffer B (see recipe)
  • Proteinase K
  • SDS
  • Viral RNA
  • Annealing buffer C (see recipe)
  • 30°, 37°, 45°, 60°, 80°, and 100°C water baths
  • Additional reagents and equipment for purifying DNA (Moore and Dowhan, )
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Literature Cited

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