Acid guanidinium thiocyanate-phenol-chloroform extraction

Phenol–chloroform extraction (abbreviated PC or PCIA, see reagents below) is a liquid–liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating DNA, RNA and protein. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a biphasic mixture. This method may take longer than a column-based system such as the silica-based purification, but has higher purity and the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short (<200 nucleotides) RNA species, such as siRNA, miRNA, gRNA and tRNA. Column methods also shear large DNA fragments, which may or may not be a problem depending on downstream applications.

It was originally devised by Piotr Chomczynski and Nicoletta Sacchi and published in 1987 (referred to as Guanidinium thiocyanate-phenol-chloroform extraction). The reagent used specifically for RNA extraction is sold by Sigma-Aldrich by the name TRI Reagent, by Invitrogen under the name TRIzol, and by Bioline as Trisure

How it works

This method relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water-saturated phenol, chloroform and a chaotropic denaturing solution (guanidinium thiocyanate) resulting in an upper aqueous phase and a lower organic phase (mainly chloroform). Nucleic acid (RNA, DNA) partitions in the aqueous phase, while protein partitions in organic phase. In a last step, RNA is recovered from the aqueous phase by precipitation with 2-propanol or ethanol. DNA will be located in the aqueous phase in the absence of guanidinium thiocyanate and thus the technique can be used for DNA purification alone.

Guanidinium thiocyanate denatures proteins, including RNases, and separates rRNA from ribosomes, while phenol, isopropanol and water are solvents with poor solubility. In the presence of chloroform or BCP (bromochloropropane), these solvents separate entirely into two phases that are recognized by their color: a clear, upper aqueous phase (containing the nucleic acids) and a bright pink lower phase (containing the proteins dissolved in phenol and the lipids dissolved in chloroform). 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 DNA.

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 (sometimes called "25:24:1"). 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 around pH 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 mixtures 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 RNase.

See also

• Column-based nucleic acid purification
• Nucleic acid methods
• Ethanol precipitation
• DNA separation by silica adsorption

External links

• Manual for TRI reagent
• Protocol on OpenWetWare
• invitrogen
• Ambion