RNA Isolation - The Proteinase K method

The isolation of RNA is more challenging than that of DNA. Samples can be easily contaminated by the ubiquitous ribonuclease, which causes RNA to be fragmented. Hence, the use of sterile glassware and chemicals of high purity is imperative. Additionally, RNA forms tight complexes with proteins. Coarse treatment is required to release RNA from these complexes. 

A common method for RNA isolation is the proteinase K method. In this method, the cells are lysed by incubation in a hypotonic solution followed by centrifugation to remove DNA and cell debris. Treatment with the proteolytic enzyme proteinase K leads to the dissociation of RNA-protein complexes and the digestion of the proteins. The digestion products are then removed by phenol/chloroform extraction and the RNA in the remaining aqueous solution is precipitated using ethanol. 

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