Single‐Step Method of Total RNA Isolation by Guanidine–Phenol Extraction


The original single‐step method is the first procedure to isolate purified total ribonucleic acid (RNA) from a variety of sources including tissues and cells from human, animal, plant, yeast, bacterial and viral origins, without the requirement of high‐speed ultracentrifugation. This method is based on liquid‐phase separation resulting in sequestration of pure RNA into the aqueous phase. RNA is precipitated from the aqueous phase, dissolved, reprecipitated and washed with alcohol before the final solubilisation step. The entire procedure can be completed in less than 4 h and it provides RNA that is suitable for many sensitive downstream applications such as RNase protection assays, northern blotting, RNA sequencing studies and reverse transcription‐polymerase chain reaction. This pioneering methodology has served as the impetus for the development of newer and improved RNA extraction methodology that now enables investigators to extract and purify RNA in less than 60 min. Some of these newer methods do not require a halogenated organic solvent, or combine the single‐step method with column purification of the RNA.

Key Concepts

  • Enzymes within living cells rapidly degrade RNA after a tissue sample is removed from the donor. To prevent RNA degradation, tissue samples that cannot be immediately processed must be rapidly frozen with dry ice or liquid nitrogen and stored frozen at −80°C until the RNA can be extracted.
  • At the time of RNA extraction, frozen cells or tissues must be immersed in the denaturing solution and rapidly homogenised before the tissue thaws in order to inactivate RNAse and avoid RNA degradation.
  • The quality of the recovered RNA is dependent on a delicate balance of salt concentration and optimal pH. Overloading the extraction solution with too much tissue or diluting the denaturing solution beyond what is specified in the protocol will impact the quality of the resulting RNA.
  • The extraction of RNA from samples that have a high buffering capacity, such as blood, plasma or tissue culture medium, requires greater care in order to maintain optimal salt balance and pH control.
  • Overdrying of the RNA pellets will degrade RNA and impede RNA solubilisation.
  • RNA should be solubilised at a concentration that will be appropriate for meaningful spectrophotometric quantitation as well as subsequent downstream molecular biology applications.
  • Enzymes that are involved in RNA degradation are ubiquitous and special care must be taken to avoid RNAse contamination during RNA solubilisation and storage.

Keywords: ribonucleic acid; single‐step method; liquid‐phase separation; total RNA isolation

Figure 1. The bioanalyser separation profile of rat liver RNA isolated by the single‐step method. The beginning of the separation is noted by the appearance of the marker (M). The capillary electrophoretic separation begins with low molecular weight transfer RNA (t‐RNA) and small RNA species, followed by the larger 18 and 28S ribosomal RNA peaks and unprocessed ribosomal precursor molecules and nuclear RNA (large RNA). Messenger RNA is diffusely spread throughout the entire separation profile and it ranges in size from 0.3 to 16 kb, but it only constitutes approximately 5% of the total RNA in the sample.


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Further Reading

Banerjee S, Smallwood A, Chambers AE and Nicolaides K (2003) Quantitative recovery of immunoreactive proteins from clinical samples following RNA and DNA isolation. BioTechniques 35: 450–456.

Blow JA, Dohm DJ, Negley DL and Mores CN (2004) Virus inactivation by nucleic acid extraction reagents. Journal of Virological Methods 119: 195–198.

Chomczynski P and Sacchi N (2006) The single‐step method of RNA isolation by acid guanidinium‐thiocyanate‐phenol‐chloroform extraction: twenty‐something years on. Nature Protocols 1: 581–585.

Copois V, Bibeau F, Bascoul‐Mollevi C, et al. (2007) Impact of RNA degradation on gene expression profiles: assessment of different methods to reliably determine RNA quality. Journal of Biotechnology 127: 549–559.

Fleige S and Pfaffl MW (2006) RNA integrity and the effect on the real‐time qRT‐PCR performance. Molecular Aspects of Medicine 27: 126–139.

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Perez‐Novo CA, Claeys C, Speleman F, et al. (2005) Impact of RNA quality on reference gene expression stability. BioTechniques 39: 52–55.

Spruessel A, Steimann G, Jung M, et al. (2004) Tissue ischemia time affects gene and protein expression patterns within minutes following surgical tumor excision. BioTechniques 36: 1030–1037.

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Wilfinger W, Mackey K and Chomczynski P (2006) Assessing the quantity, purity and integrity of RNA and DNA following nucleic acid purification. In: Kieleczawa J (ed) DNA Sequencing II Optimizing Preparation and Cleanup, pp. 291–312. Sudbury, MA: Jones and Bartlett Publishers.

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Chomczynski, Piotr, Wilfinger, William, and Mackey, Karol(Oct 2017) Single‐Step Method of Total RNA Isolation by Guanidine–Phenol Extraction. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0003799.pub3]