Preparation of RNA from Tissues and Cells

Randall Ribaudo1, Michael Gilman2, Robert E. Kingston3, Piotr Choczynski4, Nicoletta Sacchi5

1 National Institute of Allergy and Infectious Disease, Bethesda, Maryland, 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 3 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 4 University of Cincinnati College of Medicine, Cincinnati, Ohio, 5 Life Technologies, Gaithersburg, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.11
DOI:  10.1002/0471142735.im1011s04
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA.

The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A+) mRNAs from total RNA.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Preparation and Analysis of RNA
  • Preparation of RNA from Tissues and Cells
  • Basic Protocol 1: Hot Phenol Extraction of RNA
  • Basic Protocol 2: Preparation of Cytoplasmic RNA from Tissue Culture Cells
  • Support Protocol 1: Removal of Contaminating DNA from an RNA Preparation
  • Basic Protocol 3: Guanidinium Method for Total RNA Preparation
  • Alternate Protocol 1: Single‐Step RNA Isolation from Cultured Cells or Tissues
  • Basic Protocol 4: Preparation of POLY(A)+ RNA
  • Reagents and Solutions
  • Commentary
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Hot Phenol Extraction of RNA

  Materials
  • Tissue or cells
  • recipeSodium acetate buffer, ice cold
  • recipePhosphate‐buffered saline (PBS; appendix 2A), ice cold or serum‐free medium, ice cold
  • 10% SDS
  • recipeSodium acetate–saturated phenol, at 60°C
  • Chloroform
  • 95% and 70% ethanol, −20°C
  • recipeTE buffer, pH 8.0 ( appendix 2A)
  • 15‐ and 50‐ml polypropylene tubes
  • Tissue homogenizer (Brinkman or Polytron type)
  • 60°C water bath

Basic Protocol 2: Preparation of Cytoplasmic RNA from Tissue Culture Cells

  Materials
  • Diethylpyrocarbonate (DEPC)
  • recipePhosphate‐buffered saline (PBS; appendix 2A), ice cold
  • recipeLysis buffer, ice‐cold
  • 20% sodium dodecyl sulfate (SDS)
  • 20 mg/ml proteinase K
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 10.1)
  • 24:1 chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2
  • 100% ethanol
  • 75% ethanol/25% 0.1 M sodium acetate, pH 5.2
  • recipeDEPC‐treated water
  • Beckman JS‐4.2 rotor (or equivalent)
  • Rubber policeman
Water and sodium acetate should be treated with DEPC to inhibit RNase activity. See reagents and solutions for instructions.CAUTION:DEPC is a suspected carcinogen and should be handled carefully.

Support Protocol 1: Removal of Contaminating DNA from an RNA Preparation

  Additional Materials
  • recipeTE buffer, pH 7.4 ( appendix 2A)
  • 100 mM MgCl 2/10 mM dithiothreitol (DTT)
  • recipe2.5 mg/ml RNase‐free DNase I
  • Placental ribonuclease inhibitor (e.g., RNAsin from Promega Biotec)
  • recipeDNase stop mix

Basic Protocol 3: Guanidinium Method for Total RNA Preparation

  Materials
  • recipePhosphate‐buffered saline (PBS; appendix 2A)
  • Guanidinium solution
  • recipe5.7 M cesium chloride (CsCl)
  • recipeTES solution
  • 3 M sodium acetate, pH 5.2
  • 100% ethanol
  • Beckman JS‐4.2 and SW‐55 rotors (or equivalents)
  • Rubber policeman
  • 6‐ml syringe with 20‐G needle
  • 13 × 51–mm silanized ( appendix 3A) and autoclaved polyallomer ultracentrifuge tube
NOTE: The following solutions should be treated with DEPC to inhibit RNase activity: sodium acetate, water, and 5.7 M CsCl (see reagents and solutions).CAUTION: DEPC is a suspected carcinogen and should be handled carefully.

Alternate Protocol 1: Single‐Step RNA Isolation from Cultured Cells or Tissues

  Additional Materials
  • recipeDenaturing solution
  • 2 M sodium acetate, pH 4
  • recipeWater‐saturated phenol
  • 49:1 (v/v) chloroform/isoamyl alcohol
  • 100% isopropanol
  • 75% ethanol (prepared with recipeDEPC‐treated water)
  • 0.5% SDS, recipeDEPC‐treated
  • Sorvall SS‐34 rotor (or equivalent)
NOTE: Carry out all steps at room temperature unless otherwise stated.

Basic Protocol 4: Preparation of POLY(A)+ RNA

  Materials
  • Diethylpyrocarbonate (DEPC)
  • recipe5 M NaOH
  • Oligo(dT) cellulose
  • recipe0.1 M NaOH
  • recipePoly(A) loading buffer
  • 10 M LiCl
  • recipeMiddle wash buffer
  • recipe2 mM EDTA/0.1% sodium dodecyl sulfate (SDS)
  • 3 M sodium acetate
  • RNase‐free recipeTE buffer
  • Silanized column ( appendix 3A)
  • Silanized SW‐55 centrifuge tubes ( appendix 3A)
  • Beckman SW‐55 rotor (or equivalent)
The following solutions should be treated with DEPC to inhibit RNase activity: water, 10 M LiCl, 3 M sodium acetate.CAUTION: DEPC is a suspected carcinogen and should be handled carefully.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aviv, H. and Leder, P. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid–cellulose. Proc. Natl. Acad. Sci. U.S.A. 69:1408‐1412.
   Berk, A. J. and Sharp, P. A. 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease‐digested hybrids Cell 12:721‐732.
   Chirgwin, J.J., Przbyla, A.E., MacDonald, R.J., and Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294.
   Chomczynski, P. 1989 Product and process for isolating RNA. U.S. Patent 4,843,155.
   Chomczynski, P. and Sacchi, N. 1987. Single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 162:156‐159.
   Cox, R.A. 1968. The use of guanidinium chloride in the isolation of nucleic acids. Methods Enzymol. 12:120‐129.
   Favoloro, J., Treisman, R., and Kamen, R. 1980. Transcription maps of polyoma virus‐specific RNA: Analysis by two‐dimensional nuclease S1 gel mapping. Meth. Enzymol 65:718‐749.
   Feramisco, J.R., Smart, J.E., Burridge, K., Helfman, D.M., and Thomas, G.P. 1982. Co‐existence of vinculin and a vinculin‐like protein of higher molecular weight in smooth muscle. J. Biol. Chem. 257:11024‐11031.
   Gallagher, S. 1989. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. A.3.9‐A.3.15. Greene Publishing and Wiley‐Interscience, New York.
   Glisin, V., Crkvenjakov, R., and Byus, C. 1973. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633.
   Moore, C.L. and Sharp, P.A. 1984. Site‐specific polyadenylation in a cell‐free reaction. Cell 36:581‐591.
   Puissant, C. and Houdebine, L.M. 1990. An improvement of the single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. BioTechniques 8:148‐149.
   Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W.J., and Goodman, H.M. 1977. Rat insulin genes: Construction of plasmids containing the coding sequences. Science 196:1313.
   Zeff, R.A., Zhoo, Y.F., Lachman, H., Borriello, F., and Nathenson, S.G. 1991. Cis and trans repression of major histocompatibility complex–class I gene expression in Abelson virus transformed murine leukemia. Blood 78:524‐532.
Key References
   Chirgwin, et al., 1979. See above.
  Describes the use of guanidinium to lyse cells.
   Chomczynski and Sacchi, 1987. See above.
  Original description of the single‐step method.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library