Homemade viral RNA isolation protocol using silica columns: A comparison of four protocols

Abstract

The Wrst technique for RNA isolation described in the literature was developed by Chirgwin and coworkers in 1979 [1]. They standardized a method for breaking cellular membranes without denaturing nucleic acids through homogenization of tissue in a mixture of guanidinium thiocyanate and -mercaptoethanol followed by an ethanol extraction or ultracentrifugation in a cesium chloride gradient. Although this was a signiWcant advance at that time, the technique consumed a lot of time and was ineYcient, hazardous, and inconsistent. With more recent isolation techniques, the quality, speed, and eYciency of this process have improved. In 1987, Chomczynski and Sacchi improved on the Chirgwin method, converting the guanidinium–hot phenol method to a single-step extraction called the AGPC (acid guanidinium–phenol–chloroform) method [2]. This modiWcation had at least two beneWts: the possibility of processing more samples simultaneously and the isolation of unbroken RNA with a higher purity and yield. Some years later, in 1993, Chomczynski published a reagent for the single-step simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples [3]. Nowadays, there are several nucleic acid isolation kits on the market based on this reagent. More recently, some commercial alternatives have been developed, but it was the development of new techniques employing silica columns for the isolation of nucleic acids