Isolation of RNA using guanidinium salts

As an alternative to phenol extraction for the isolation of RNA, homogenization in guanidine hydrochloride solutions is an inexpensive and convenient method. For those samples with very high RNase content or for irreplaceable samples with unknown RNase content, guanidinium thiocyanate becomes the deproteinizing agent of choice. Efficient protein denaturation with consequent elimination of nucleolytic damage to the RNA requires rapid and complete homogenization. For instance, homogenization of a rat pancreas in guanidinium thiocyanate solution by means of a motorized pestle results in extensively degraded RNA, which is avoided by the use of an apparatus such as a Tissumizer or Polytron. Frozen tissue should be pulverized in liquid nitrogen, then homogenized immediately (without thawing) after adding to the guanidinium solution. Once the RNA is dispersed in the guanidinium solution, steps (1) and (2) have been completed, and only step (3) remains: physical separation of the RNA from the other macromolecular components of the homogenate. A number of separation methods have been described in the literature, and these can be divided into two main categories: selective precipitation based on solubility, and selective sedimentation (by ultracentrifugation) based on buoyant density.