Guanidine Methods for Total RNA Preparation

Robert E. Kingston1, Piotr Chomczynski2, Nicoletta Sacchi3

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 2 University of Cincinnati College of Medicine, Cincinnati, Ohio, 3 Laboratory of Molecular Oncology National Cancer Institute, Frederick, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.2
DOI:  10.1002/0471142727.mb0402s36
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Three different methods for RNA preparation using guanidine are presented in this unit ‐‐ a single‐step isolation method employing liquid‐phase separation to selectively extract total RNA from tissues and cultured cells and two methods that rely on a CsCl step gradient to isolate total RNA.

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

Table of Contents

  • Basic Protocol 1: Single‐Step RNA Isolation from Cultured Cells or Tissues
  • Alternate Protocol 1: CsCl Purification of RNA from Cultured Cells
  • Alternate Protocol 2: CsCl Purification of RNA from Tissue
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

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

  Materials
  • recipeDenaturing solution (see recipe)
  • recipe2 M sodium acetate, pH 4 (see recipe)
  • recipeWater‐saturated phenol (see recipe)
  • 49:1 (v/v) chloroform/isoamyl alcohol or bromochloropropane
  • 100% isopropanol
  • 75% ethanol (prepared with DEPC‐treated water; unit 4.1)
  • DEPC‐treated water (unit 4.1) or recipefreshly deionized formamide (see recipe)
  • Glass Teflon homogenizer
  • 5‐ml polypropylene centrifuge tube
  • Sorvall SS‐34 rotor (or equivalent)
CAUTION: Phenol is a poison and causes burns. When handling phenol, use gloves and eye protection.NOTE: Carry out all steps at room temperature unless otherwise stated.

Alternate Protocol 1: CsCl Purification of RNA from Cultured Cells

  • Phosphate‐buffered saline (PBS; appendix 22)
  • recipeGuanidine solution (see recipe)
  • recipe5.7 M cesium chloride (CsCl), DEPC‐treated (see recipe)
  • recipeTES solution (see recipe)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 100% ethanol
  • Rubber policeman
  • 6‐ml syringe with 20‐G needle
  • Beckman JS‐4.2 and SW 55 rotors (or equivalents)
  • 13 × 51–mm silanized ( appendix 3B) and autoclaved polyallomer ultracentrifuge tube
  • Additional reagents and equipment for quantitating RNA ( appendix 3D)
CAUTION: DEPC is a suspected carcinogen and should be handled carefully.NOTE: The following solutions should be treated with DEPC to inhibit RNase activity: sodium acetate, water, and 5.7 M CsCl (unit 4.1).NOTE: Carry out steps 1 to at room temperature.

Alternate Protocol 2: CsCl Purification of RNA from Tissue

  • Liquid nitrogen
  • recipeTissue guanidine solution (see recipe)
  • 20% (w/v) N‐lauroylsarcosine (Sarkosyl)
  • Cesium chloride (CsCl)
  • recipeTissue resuspension solution (see recipe)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 24:1 chloroform/isoamyl alcohol
  • Tissuemizer
  • Sorvall SS‐34 and Beckman SW 28 rotors (or equivalents)
  • SW 28 polyallomer tube silanized ( appendix 3B) and autoclaved
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   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. 1992. Solubilization in formamide protects RNA from degradation. Nucl. Acids Res. 20:3791‐3792.
   Chomczynski, P. and Mackey, K. 1995. Substitution of chloroform by bromochloropropane in the single‐step method of RNA isolation. Anal. Biochem. 225:163‐164.
   Chomczynski, P. and Sacchi, N. 1987. Single‐step method of RNA isolation by acid guanidine thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 162:156‐159.
   Cox, R.A. 1968. The use of guanidine chloride in the isolation of nucleic acids. Methods Enzymol. 12:120‐129.
   Ferimisco, 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.
   Glisin, V., Crkvenjakov, R., and Byus, C. 1974. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633.
   Herrin, D.L. and Schmidt, G.W. 1988. Rapid, reversible staining of northern blots prior to hybridization. BioTechniques 6:196‐200.
   Puissant, C. and Houdebine, L.M. 1990. An improvement of the single‐step method of RNA isolation by acid guanidine 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.
   Willfinger, W.W., Mackey, K. and Chomczynski, P. 1996. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Submitted for publication.
Key References
   Chirgwin et al., 1979. See above.
  Describes the use of guanidine to lyse cells.
   Chomczynski and Sacchi 1978. See above.
  Original description of the single‐step method.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library