Purely RNA

Fundamental laboratory techniques such as Northern blot analysis, RNA protection assays, in situ hybridization, and reverse transcriptase-PCR (RT-PCR) require high-quality, highly purified RNA samples. Preparing such samples is often laborious at best, because RNAses-both stable and omnipresent-can easily degrade the samples. In the past, RNA work was often left to dedicated labs, with dedicated work areas, equipment, and some very meticulous workers. Fortunately, the technology continues to evo

Written byDeborah Stull

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The first step to RNA recovery is cell lysis, which depends on the denaturation of the proteins that comprise the cellular membrane. In 1979 John Chirgwin and colleagues devised a method for disrupting cellular membranes without denaturing nucleic acids. They homogenized tissue in guanidinium thiocyanate, a protein denaturant, and b-mercaptoethanol, a reducing agent.¹ These scientists then isolated and purified the cellular RNA through either ethanol extraction or ultracentrifugation across a cesium chloride gradient. This technique was the first to allow researchers to isolate RNA specifically, but it had a number of drawbacks-it was lengthy, inefficient, hazardous, and inconsistent.

In 1987 Piotr Chomczynski and Nicoletta Sacchi improved on this method by combining the extraction procedures into one step through the use of a mixture of guanidinium thiocyanate and phenol-chloroform.2 This modification had at least two tangible benefits. First, it reduced the length of the RNA-isolation step, allowing researchers to increase the ...

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