Isolation of RNA using guanidinium salts

R. J. MacDonald, G. H. Swift, A. E. Przybyla, J. M. Chirgwin

Research output: Chapter in Book/Report/Conference proceedingChapter

508 Citations (Scopus)

Abstract

As an alternative to phenol extraction for the isolation of RNA, homogenization in guanidine hydrochloride solutions is an inexpensive and convenient method. For those samples with very high RNase content or for irreplaceable samples with unknown RNase content, guanidinium thiocyanate becomes the deproteinizing agent of choice. Efficient protein denaturation with consequent elimination of nucleolytic damage to the RNA requires rapid and complete homogenization. For instance, homogenization of a rat pancreas in guanidinium thiocyanate solution by means of a motorized pestle results in extensively degraded RNA, which is avoided by the use of an apparatus such as a Tissumizer or Polytron. Frozen tissue should be pulverized in liquid nitrogen, then homogenized immediately (without thawing) after adding to the guanidinium solution. Once the RNA is dispersed in the guanidinium solution, steps (1) and (2) have been completed, and only step (3) remains: physical separation of the RNA from the other macromolecular components of the homogenate. A number of separation methods have been described in the literature, and these can be divided into two main categories: selective precipitation based on solubility, and selective sedimentation (by ultracentrifugation) based on buoyant density.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
Pages219-226
Number of pages8
Volume152
StatePublished - 1987

Fingerprint

Guanidine
Salts
RNA
Ribonucleases
Protein Denaturation
Denaturation
Thawing
Ultracentrifugation
Liquid nitrogen
Phenol
Sedimentation
Solubility
Rats
Pancreas
Nitrogen
Tissue
Proteins
guanidine thiocyanate

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

MacDonald, R. J., Swift, G. H., Przybyla, A. E., & Chirgwin, J. M. (1987). Isolation of RNA using guanidinium salts. In Methods in Enzymology (Vol. 152, pp. 219-226)

Isolation of RNA using guanidinium salts. / MacDonald, R. J.; Swift, G. H.; Przybyla, A. E.; Chirgwin, J. M.

Methods in Enzymology. Vol. 152 1987. p. 219-226.

Research output: Chapter in Book/Report/Conference proceedingChapter

MacDonald, RJ, Swift, GH, Przybyla, AE & Chirgwin, JM 1987, Isolation of RNA using guanidinium salts. in Methods in Enzymology. vol. 152, pp. 219-226.
MacDonald RJ, Swift GH, Przybyla AE, Chirgwin JM. Isolation of RNA using guanidinium salts. In Methods in Enzymology. Vol. 152. 1987. p. 219-226
MacDonald, R. J. ; Swift, G. H. ; Przybyla, A. E. ; Chirgwin, J. M. / Isolation of RNA using guanidinium salts. Methods in Enzymology. Vol. 152 1987. pp. 219-226
@inbook{41e59e95033b485c9242a8e43ef715cc,
title = "Isolation of RNA using guanidinium salts",
abstract = "As an alternative to phenol extraction for the isolation of RNA, homogenization in guanidine hydrochloride solutions is an inexpensive and convenient method. For those samples with very high RNase content or for irreplaceable samples with unknown RNase content, guanidinium thiocyanate becomes the deproteinizing agent of choice. Efficient protein denaturation with consequent elimination of nucleolytic damage to the RNA requires rapid and complete homogenization. For instance, homogenization of a rat pancreas in guanidinium thiocyanate solution by means of a motorized pestle results in extensively degraded RNA, which is avoided by the use of an apparatus such as a Tissumizer or Polytron. Frozen tissue should be pulverized in liquid nitrogen, then homogenized immediately (without thawing) after adding to the guanidinium solution. Once the RNA is dispersed in the guanidinium solution, steps (1) and (2) have been completed, and only step (3) remains: physical separation of the RNA from the other macromolecular components of the homogenate. A number of separation methods have been described in the literature, and these can be divided into two main categories: selective precipitation based on solubility, and selective sedimentation (by ultracentrifugation) based on buoyant density.",
author = "MacDonald, {R. J.} and Swift, {G. H.} and Przybyla, {A. E.} and Chirgwin, {J. M.}",
year = "1987",
language = "English (US)",
volume = "152",
pages = "219--226",
booktitle = "Methods in Enzymology",

}

TY - CHAP

T1 - Isolation of RNA using guanidinium salts

AU - MacDonald, R. J.

AU - Swift, G. H.

AU - Przybyla, A. E.

AU - Chirgwin, J. M.

PY - 1987

Y1 - 1987

N2 - As an alternative to phenol extraction for the isolation of RNA, homogenization in guanidine hydrochloride solutions is an inexpensive and convenient method. For those samples with very high RNase content or for irreplaceable samples with unknown RNase content, guanidinium thiocyanate becomes the deproteinizing agent of choice. Efficient protein denaturation with consequent elimination of nucleolytic damage to the RNA requires rapid and complete homogenization. For instance, homogenization of a rat pancreas in guanidinium thiocyanate solution by means of a motorized pestle results in extensively degraded RNA, which is avoided by the use of an apparatus such as a Tissumizer or Polytron. Frozen tissue should be pulverized in liquid nitrogen, then homogenized immediately (without thawing) after adding to the guanidinium solution. Once the RNA is dispersed in the guanidinium solution, steps (1) and (2) have been completed, and only step (3) remains: physical separation of the RNA from the other macromolecular components of the homogenate. A number of separation methods have been described in the literature, and these can be divided into two main categories: selective precipitation based on solubility, and selective sedimentation (by ultracentrifugation) based on buoyant density.

AB - As an alternative to phenol extraction for the isolation of RNA, homogenization in guanidine hydrochloride solutions is an inexpensive and convenient method. For those samples with very high RNase content or for irreplaceable samples with unknown RNase content, guanidinium thiocyanate becomes the deproteinizing agent of choice. Efficient protein denaturation with consequent elimination of nucleolytic damage to the RNA requires rapid and complete homogenization. For instance, homogenization of a rat pancreas in guanidinium thiocyanate solution by means of a motorized pestle results in extensively degraded RNA, which is avoided by the use of an apparatus such as a Tissumizer or Polytron. Frozen tissue should be pulverized in liquid nitrogen, then homogenized immediately (without thawing) after adding to the guanidinium solution. Once the RNA is dispersed in the guanidinium solution, steps (1) and (2) have been completed, and only step (3) remains: physical separation of the RNA from the other macromolecular components of the homogenate. A number of separation methods have been described in the literature, and these can be divided into two main categories: selective precipitation based on solubility, and selective sedimentation (by ultracentrifugation) based on buoyant density.

UR - http://www.scopus.com/inward/record.url?scp=0023544693&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023544693&partnerID=8YFLogxK

M3 - Chapter

VL - 152

SP - 219

EP - 226

BT - Methods in Enzymology

ER -