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The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on

Abstract

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.

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Figure 1: Electrophoresis of RNAs isolated by the single-step method.

References

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Acknowledgements

We thank G. Bistulfi (Roswell Park Cancer Institute) for re-editing the original protocol in this new format.

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Correspondence to Nicoletta Sacchi.

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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.).

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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

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