Identification of Newly Transcribed RNA

Michael E. Greenberg1, Timothy P. Bender2

1 Harvard Medical School, Boston, Massachusetts, 2 University of Virginia, Charlottesville, Virginia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.10
DOI:  10.1002/0471142727.mb0410s78
Online Posting Date:  April, 2007
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Abstract

Newly transcribed RNA can be identified using the nuclear runoff transcription assay. In this assay, isolated nuclei, free of membranes and cytoplasmic debris, are used in an in vitro transcription reaction in the presence of 32P‐labeled UTP. The labeled RNA can then be hybridized to cDNAs immobilized on nitrocellulose. This unit describes three methods for isolating suitable nuclei by detergent lysis, Dounce homogenization, and centrifugation on a sucrose gradient. Two Support Protocols describe the preparation of nitrocellulose filter strips containing double‐stranded and single‐stranded DNAs, which are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.

Keywords: nuclear runoff transcription assay; isolated nuclei

     
 
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Table of Contents

  • Basic Protocol 1: Nuclear Runoff Transcription in Mammalian Cells
  • Alternate Protocol 1: Isolation of Nuclei by Dounce Homogenization
  • Alternate Protocol 2: Isolation of Nuclei by Sucrose Gradient Centrifugation
  • Support Protocol 1: Preparation of Nitrocellulose Filters with Double‐Stranded Targets
  • Support Protocol 2: Preparation of Nitocellulose Filters with Single‐Stranded Targets
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Nuclear Runoff Transcription in Mammalian Cells

  Materials
  • Cultures of mammalian cells ( appendix 3F) or freshly isolated lymphoid cells
  • Phosphate‐buffered saline (PBS; appendix 22), made fresh and ice cold
  • NP‐40 lysis buffer A (see recipe)
  • Glycerol storage buffer (see recipe), ice cold
  • 2× reaction buffer with and without nucleotides (see recipe)
  • 10 mCi/ml [α‐32P]UTP (760 Ci/mmol)
  • 1 mg/ml DNase I (RNase‐free; see recipe)
  • HSB buffer (see recipe)
  • SDS/Tris buffer (see recipe)
  • 20 mg/ml proteinase K
  • 25:24:1 (v/v/v) buffered phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 10% (v/v) trichloroacetic acid (TCA)/60 mM sodium pyrophosphate
  • 10 mg/ml tRNA (see recipe)
  • 5% (v/v) TCA/30 mM sodium pyrophosphate
  • DNase I buffer (see recipe)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 20% (w/v) SDS
  • Elution buffer (see recipe)
  • 1 M NaOH
  • 1 M HEPES (free acid)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 100% ethanol
  • TES solution (see recipe)
  • TES/NaCl solution (see recipe)
  • cDNA plasmid(s) immobilized on nitrocellulose membrane ( protocol 4 or 2)
  • 2× SSC ( appendix 22)
  • 10 mg/ml heat‐inactivated RNase A (see recipe)
  • Rubber policeman
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Beckman JS‐4.2 and JA‐20 rotors or equivalent
  • 30° and 65°C shaking water baths
  • 42° and 65°C water baths
  • 0.45‐µm HA filters (Millipore)
  • 30‐ml Corex tube, silanized ( appendix 22)
  • Whatman GF/F glass fiber filters
  • 5‐ml plastic scintillation vials
  • Whatman 3MM filter paper
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3F), phenol/chloroform extraction and ethanol precipitation of nucleic acids (unit 2.1), hybridization to RNA slot blots (unit 4.9), and autoradiography ( appendix 3A)
NOTE: Keep cells and nuclei on ice until the nuclei are frozen.

Alternate Protocol 1: Isolation of Nuclei by Dounce Homogenization

  • Lysis buffer (see recipe), ice cold
  • NP‐40 lysis buffer B (see recipe)
  • Glycerol storage buffer (see recipe), ice cold
  • Dounce homogenizer with type B pestle, ice cold
  • 1.5‐ml microcentrifuge tubes, chilled on dry ice
NOTE: Keep cells and nuclei on ice until the nuclei are frozen.

Alternate Protocol 2: Isolation of Nuclei by Sucrose Gradient Centrifugation

  • Sucrose buffer I (see recipe), ice cold
  • Sucrose buffer II (see recipe)
  • Dounce homogenizer with B pestle, ice cold
  • Polyallomer centrifuge tubes (9/16 × 3 3/4 in., Beckman) for SW 40.1 rotor
  • Ultracentrifuge and SW 40.1 rotor or equivalent
  • 1.5‐ml microcentrifuge tubes, chilled on dry ice
NOTE: Keep cells and nuclei on ice until the nuclei are frozen.

Support Protocol 1: Preparation of Nitrocellulose Filters with Double‐Stranded Targets

  Materials
  • cDNA plasmid
  • 1 M NaOH
  • 6× SSC ( appendix 22)
  • 0.45‐µm nitrocellulose membrane
  • Slot blot apparatus
  • 80°C vacuum oven
  • Additional reagents and equipment for restriction endonuclease digestion (unit 3.1) and RNA slot blots (unit 4.9)
NOTE: Wear gloves and handle membranes with blunt‐ended forceps.

Support Protocol 2: Preparation of Nitocellulose Filters with Single‐Stranded Targets

  Materials
  • Target DNA prepared as M13 ssDNA clone (unit 7.3)
  • 1 N NaOH
  • 2 M sodium acetate buffer, pH 7 ( appendix 22)
  • 10× SSC ( appendix 22)
  • 65°C incubator
  • 0.45‐µm nitrocellulose membrane
  • Slot blot apparatus
  • 80°C vacuum oven
  • Additional reagents and equipment for RNA slot blots (unit 4.9)
NOTE: Wear gloves and handle membranes with blunt‐ended forceps.
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Figures

Videos

Literature Cited

   Marzluff, W.F. 1978. Transcription of RNA in isolated nuclei. Methods Cell Biol. 19:317‐331.
   Marzluff, W.F. and Huang, R.C.C. 1985. Transcription and Translation: A Practical Approach (B.D. Hames and S.J. Higgins, eds.) pp. 89‐129. IRL Press, Oxford.
Key References
   Greenberg, M.E. and Ziff, E.B. 1984. Stimulation of 3T3 cells induces transcription of the c‐fos protooncogene. Nature. 311:433‐438.
  Describes the use of the nuclear runoff technique to measure the transcription rates of numerous genes as a function of a specific change in the cellular environment.
   Groudine, M., Peretz, M., and Weintraub, H. 1981. Transcriptional regulation of hemoglobin switching on chicken embryos. Mol. Cell. Biol. 1:281‐288.
  Describes the nuclear runoff transcription assay upon which the is based.
   Marzluff and Huang 1985. See above.
  Contains a great deal of useful information on the nuclear runoff transcription assay and describes in detail problems encountered in isolating the nuclei.
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