This method relies on phase separation by centrifugation of a mixture of the aqueous sample and a solution containing water-saturated phenol and chloroform, resulting in an upper aqueous phase and a lower organic phase. Guanidinium thiocyanate, a chaotropic agent, is added to the organic phase to aid in the denaturation of proteins. The nucleic acids partition into the aqueous phase, while protein partitions into the organic phase. The pH of the mixture determines which nucleic acids get purified. Under acidic conditions, DNA partitions into the organic phase while RNA remains in the aqueous phase. Under neutral conditions, both DNA and RNA partition into the aqueous phase. In a last step, the nucleic acids are recovered from the aqueous phase by precipitation with 2-propanol. The 2-propanol is then washed with ethanol and the pellet briefly air-dried and dissolved in TE buffer or RNAse free water. Guanidinium thiocyanate denatures proteins, including RNases, and separates rRNA from ribosomal proteins, while phenol, isopropanol and water are solvents with poor solubility. In the presence of chloroform or BCP, these solvents separate entirely into two phases that are recognized by their color: a clear, upper aqueous phase and a lower phase. Other denaturing chemicals such as 2-mercaptoethanol and sarcosine may also be used. The major downside is that phenol and chloroform are both hazardous and inconvenient materials, and the extraction is often laborious, so in recent years many companies now offer alternative ways to isolate RNA.
Reagents
Phenol: The phenol used for biochemistry comes as a water-saturated solution with Tris buffer, as a Tris-buffered 50% phenol, 50% chloroform solution, or as a Tris-buffered 50% phenol, 48% chloroform, 2% isoamyl alcohol solution. Phenol is naturally somewhat water-soluble, and gives a fuzzy interface, which is sharpened by the presence of chloroform, and the isoamyl alcohol reduces foaming. Most solutions also have an antioxidant, as oxidized phenol damages the nucleic acids. For RNA purification, the pH is kept at around 4, which retains RNA in the aqueous phase preferentially. For DNA purification, the pH is usually near 7, at which point all nucleic acids are found in the aqueous phase.
Chloroform: Chloroform is stabilized with small quantities of amylene or ethanol, because exposure of pure chloroform to oxygen and ultraviolet light produces phosgene gas. Some chloroform solutions come as pre-made a 96% chloroform, 4% isoamyl alcohol mixture that can be mixed with an equal volume of phenol to obtain the 25:24:1 solution.
Isoamyl alcohol: Isoamyl alcohol may reduce foaming and ensure deactivation of RNases.
