Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. & Rutter, W.J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294–5299 (1979).
Cox, R.A. The use of guanidine chloride in the isolation of nucleic acids. Methods Enzymol. 12, 120–129 (1968).
Chomczynski, P. & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159 (1987).
Puissant, C. & Houdebine, L.M. An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Biotechniques 8, 148–149 (1990).
Chomczynski, P. Solubilization in formamide protects RNA from degradation. Nucleic Acids Res. 20, 3791–3792 (1992).
Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning. A Laboratory Manual. Second Edition. (1989).
Wilfinger, W. W., Mackey, K. & Chomczynski, P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22, 474–6, 478–81 (1997).
We thank G. Bistulfi (Roswell Park Cancer Institute) for re-editing the original protocol in this new format.
The original method described in this article (Chomczynski, P. & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162, 156–159 (1987)) is covered by US patent 4,843,155 (Chomczynski, P., Product and process for isolating RNA, 1989.).
About this article
Cite this article
Chomczynski, P., Sacchi, N. The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat Protoc 1, 581–585 (2006). https://doi.org/10.1038/nprot.2006.83
An efficient overexpression method for studying genes in Ricinus that transport vectorized agrochemicals
Plant Methods (2022)
Communications Biology (2022)
Journal of Human Genetics (2022)
Nature Communications (2022)
Scientific Reports (2022)