Northern Blot Analysis of RNA

Marcelle Bergeron1, Jari Honkaniemi2, Frank R. Sharp3

1 Lilly Research Laboratories, Indianapolis, Indiana, 2 Tampere University Hospital, Tampere, Finland, 3 University of Cincinnati, Cincinnati, Ohio
Publication Name:  Current Protocols in Toxicology
Unit Number:  Appendix 3E
DOI:  10.1002/0471140856.txa03es07
Online Posting Date:  May, 2001
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Abstract

This unit describes the processes of extracting RNA from tissues and analyzing it by northern blot hybridization to probe the expression of a particular gene at the transcriptional level.

     
 
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Table of Contents

  • Basic Protocol 1: Northern Blot Hybridization
  • Support Protocol 1: Total RNA Extraction
  • Support Protocol 2: Radiolabeling an Oligonucleotide Probe
  • Support Protocol 3: Stripping and Reprobing the Membrane
  • Reagents and Solutions
  • Figures
     
 
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Materials

Basic Protocol 1: Northern Blot Hybridization

  Materials
  • Ultrapure agarose, low‐melting temperature (e.g., Life Technologies, Bio‐Rad, Sigma, or Fisher)
  • DEPC‐treated H 2O (unit 2.9)
  • 10× and 1× MOPS (see recipe)
  • 37% formaldehyde (Sigma)
  • Purified RNA (20 to 40 µg total or 4 to 10 µg mRNA; see protocol 2)
  • ≥99.5% formamide, deionized (Sigma)
  • RNA loading dye (see recipe)
  • 10 mg/ml ethidium bromide (optional; see recipe)
  • Nylon membrane, positively charged (Hybond from Amersham Pharmacia Biotech or Nytran SuPerCharge from Schleicher & Schuell)
  • 20× and 2× SSC (unit 2.9)
  • 0.02% (w/v) methylene blue RNA staining solution (unit 2.9)
  • 20× SSPE (unit 2.9)
  • 20% (w/v) SDS ( appendix 2A)
  • 100× Denhardt's solution (unit 2.9)
  • 10 mg/ml heat‐denatured salmon sperm DNA (Life Technologies)
  • 50% (w/v) dextran sulfate (unit 2.9)
  • 100 cpm/µg [α‐32P]dATP‐labeled oligonucleotide probe (see protocol 3)
  • 1× and 2× SSPE/0.1% SDS
  • 1× STE buffer (see recipe)
  • Horizontal gel electrophoresis system with casting box and Teflon combs with 3‐ to 4‐mm teeth (e.g., Bio‐Rad, Amersham Pharmacia Biotech, Owl, Shadel) and appropriate power supply
  • Microcentrifuge tubes, RNase‐free
  • UV transilluminator, ruler, and camera (optional)
  • Elevated support (e.g., empty pipet tip box)
  • Whatman 3 MM papers
  • 10‐ to 15‐cm stack of paper towels
  • Small weight (∼500 g) or light‐weight cover
  • UV Cross‐linker (Stratagene)
  • Pencil or fluorescent pen (optional)
  • Hybridization oven with glass hybridization bottle (Hybaid) or
  • 42°C water bath and sealable bag
  • Geiger counter
  • Autoradiographic cassette, light‐tight with intensifying screen
  • X‐ray film (e.g., X‐Omat AR5 or BioMax MS from Kodak)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1A).CAUTION: Radiolabeled probes are hazardous; see appendix 1A for guidelines on handling, storage, and disposal. To protect from radioactivity and RNase contamination as well as finger smudges, always wear clean, powder‐free gloves when working with RNA and radioactive substances. Do not reuse gloves.

Support Protocol 1: Total RNA Extraction

  • Tissue
  • Trizol Reagent (Life Technologies) or TriReagent (Sigma) or RNA STAT‐60 (Tel‐Test) or Ultraspec (Biotech Labs)
  • Chloroform
  • 5:1 (v/v) acid phenol/chloroform, pH 4.7 (Ambion)
  • Isopropyl alcohol
  • 75% and 100% ethanol
  • dH 2O, sterile, non‐DEPC treated, RNase/DNase‐ and pyrogen‐free (Ambion or Life Technologies)
  • 40 U/µl RNasin (Promega)
  • 1 M Tris⋅Cl, pH 7.5, RNase‐free (Sigma, Ambion, Life Technologies)
  • 50 mM MgCl 2, RNase‐free (Sigma, Ambion, Life Technologies)
  • 10 U/µl DNase, RNase free (Life Technologies, Roche Molecular Biochemical, Ambion)
  • 3 M sodium acetate solution, pH 5.2, RNase‐free (Sigma, Ambion)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Polytron or Dounce homogenizer
  • Pipet tips, RNase‐free (Fischer, Ambion, Promega)
NOTE: Because Tris⋅Cl, MgCl 2, and sodium acetate solution listed here are added directly to the RNA preparation, we recommend that they be absolutely RNase‐free. Because of the small volumes used in this protocol, we recommend a commercial supplier as the source for these solutions.CAUTION: Guanidine isothiocyanate and phenol are highly toxic. The use of gloves, adequate ventilation, and special disposal methods are required.

Support Protocol 2: Radiolabeling an Oligonucleotide Probe

  • 25 to 30 U/µl terminal deoxynucleotidyl transferase (TdT) and 5× TdT buffer (e.g., Life Technologies, Roche Molecular Biochemicals)
  • 100 ng/µl antisense single‐stranded oligonucleotide probe in H 2O
  • 6000 Ci/mmol [α‐32P]dATP (20 mCi/ml; 3.33 nmol/ml)
  • Scintillant solution for aqueous samples (ReadySafe from Beckman or Aquasol from Packard)
  • 1× STE (see recipe), sterile
  • 37°C heating block
  • Sephadex columns: DNase/RNase‐free STE Mirco Select‐D G‐25 Spin Column (5 Prime → 3 Prime) or NucTrap Push Column (Stratagene)
  • Scintillation counter and appropriate vials
  • β‐shield column holder (Stratogene)
  • 10‐ to 20‐ml syringes
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1A).CAUTION: When working with a radioactive isotope such as 32P, strict radiation safety precautions should be taken to avoid exposure to harmful radioactivity and to prevent contamination of nonradioactive instruments and laboratory areas (also see appendix 1A). Always wear powder‐free gloves when working with RNA and radioactivity. Do not reuse gloves.

Support Protocol 3: Stripping and Reprobing the Membrane

  • Radioactive membrane to be stripped (see protocol 1)
  • 0.01× SSPE (unit 2.9) containing 0.1% (w/v) SDS ( appendix 2A)
  • Formamide, deionized, minimum 99.5% (Sigma)
  • Secondary radioactive probe (see protocol 3)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1A).CAUTION: To protect from radioactivity and RNase contamination as well as fingerprint smudges, always wear clean, powder‐free gloves when working with RNA and radioisotopes. Do not reuse gloves.
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Figures

Videos

Literature Cited

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