Message in a test tube

Eisenstein, Michael

doi:10.1038/nmeth0206-148

Classic Protocol
Published: February 2006

Message in a test tube

Michael Eisenstein

Nature Methods volume 3, page 148 (2006)Cite this article

918 Accesses
5 Altmetric
Metrics details

Access through your institution

Buy or subscribe

People have been working with RNA for the better part of a century now. However, the earliest RNA preparation techniques were often inefficient and unreliable—involving boiling, extreme alkaline conditions or chemical denaturants—and thus often impeded progress toward better understanding of the molecule itself. One important challenge involved keeping RNA intact in the presence of ribonuclease; as early as 1920, scientists were aware that biological tissues often contain high concentrations of this robust and efficient enzyme¹, and the successful analysis of RNA depends greatly on the extent to which one can effectively protect it from degradation. Today, of course, a variety of single-solution systems are available for rapid RNA preparation, solutions that are typically based on the integration of two different classical nucleic acid preparation methodologies.

One of these methods, the now-venerable phenol-chloroform extraction, is actually the culmination of decades of working with the two reagents independently. Chloroform first came onto the scene for nucleic acid work in the 1930s, achieving 'fame' in the hands of M.G. Sevag, who observed that chloroform and protein seem to enter into an interaction in solution that excludes nucleic acids. By adding a small fraction of isoamyl alcohol to prevent foaming during the mixing process, Sevag developed a simple method for phase-dependent separation of nucleic acids from crude cell extracts²—and the resulting chloroform–isoamyl alcohol mixture became known as 'Sevag' as a result. Phenol came into use years later, when it was demonstrated how effectively phenol solutions dissolve organic tissue, while also rendering RNases inactive; the resulting solution also separates into phases, from which RNA can be readily separated from both DNA and proteins³. Klaus Scherrer and James Darnell further refined this approach with their paper describing the simple 'hot phenol' extraction of RNA from HeLa cells⁴, which would remain a favored approach for years to come.

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

Purchase on SpringerLink
Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

References

Jones, W. Am. J. Physiol. 52, 203–207 (1920).
Article CAS Google Scholar
Sevag, M.G. et al. J. Biol. Chem. 124, 425–435 (1938).
CAS Google Scholar
Kirby, K.S. Biochem. J. 64, 405–408 (1956).
Article CAS Google Scholar
Scherrer, K. & Darnell, J.E. Biochem. Biophys. Res. Commun. 7, 486–490 (1962).
Article CAS Google Scholar
Volkin, E. & Carter, C.E. U.S. Atomic Energy Commission, ORNL-587, 1 March 1950.
Grinnan, E.L. & Mosher, W.A. J. Biol. Chem. 191, 719–726 (1951).
CAS PubMed Google Scholar
Chirgwin, J.M. et al. Biochemistry 18, 5294–5299 (1979).
Article CAS Google Scholar
Chomczynski, P. Curr. Contents 6 September 1993.
Feramisco, J.R. et al. in Molecular Cloning (Maniatis, T. et al., eds.) 194–195 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, 1982).
Google Scholar
Chomczynski, P. & Sacchi, N. Anal. Biochem. 162, 156–159 (1987).
Article CAS Google Scholar