Guanidium thiocyanate-phenol-chloroform extraction

A common method used to isolate total RNA is guanidium thiocyanate-phenol-chloroform extraction. This method utilizes a chemical called TRIreagent (or TRIzol), which contains guanidinium isothiocyanate, a powerful protein denaturant, and phenol, an organic solvent. The TRIreagent buffer maintains the RNAs integrity, disrupts cells and denatures proteins. This method is based on the research of Piotr Chomczynski and Nicoletta Sacchi in 1987 & 2006. It takes slightly more time than column-based methods like RNAeasy (Qiagen), but it can handle larger amounts of tissue or cells and therefore yields more RNA.

The main steps of RNA isolation are as follows:

• Cell lysis and disruption of cellular structures.
• Separation of the RNA from cell debris.
• Purification of the RNA from the DNA and proteins.
• Precipitation of RNA.
• Wash and re-suspension of the RNA.

The lysate consists of RNA, DNA, proteins and cellular debris. Centrifugation can be used to separate the macromolecules (proteins, RNA and DNA) from the cell debris. Phase separation of the sample will occur when chloroform is added to the sample, based on the different densities. In addition, the pH will determine the separation of the nucleic acids and proteins. The polar RNA will remain in the upper polar phase, DNA will accumulate in the interphase and the denatured proteins will dissolve in the lower organic phase.

The RNA from the aqueous phase is precipitated by adding isopropanol, which precipitates polar molecules like RNAs and salts. The salts can be washed away with 75% ethanol to obtain pure RNA. Finally the RNA pellet is dried and re-suspended using RNAse free water as a buffer.

After RNA isolation the concentration and purity of the RNA needs to be assesed. Also the quality should be inspected before further analysis.