Guanidinium thiocyanate-phenol-chloroform extraction

Guanidinium thiocyanate-phenol-chloroform extraction is an extraction method for isolating RNA, DNA and protein. This method is widely used in molecular biology, and is often referred to as the TRIzol method, after the name of the Invitrogen product. It was originally devised by Piotr Chomczynski and Nicoletta Sacchi and published in 1987. This method relies on phase separation upon centrifugation of a mix of water-saturated phenol, chloroform and a chaotropic denaturing solution (guanidinium) into an aqueous phase and an organic phase (phenol). Nearly all of the RNA collects into the aqueous phase, while the DNA and protein collect in the interphase and the organic phase. This method takes longer than a column-based system such as the RNeasy column (Qiagen), but has the major advantage that no RNA is lost. A column typically passes through RNA transcripts shorter than 200 bp, including many types of noncoding RNAs including siRNA and miRNA, hot research topics in modern molecular biology. The name of the protocol derives from the chemicals used: The proteins, including RNase, are denatured by guanidinium thiocyanate which is strong enough to separate rRNA from ribosomes. Other denaturing chemicals such as 2-mercaptoethanol and sarcosine are often present. Phenol, isopropanol and water are the solvents that mix poorly, and, in the presence of chloroform or BCP (bromochloropropane), separate entirely into two phases that are easily recognized by their color: the clear, upper water phase and the bright pink lower phase.