inactivate RNases. The lysate is then processed in one of several ways to purify the RNa away from protein, genomic DNA, and other cellular components. a brief description of each method along with the time and effort involved, the quality of rna obtained, and the scalability of the procedures follow Guanidium-Cesium Chloride Method Slow, laborious procedure, but RNA is squeaky clean; unsuitable for large sample numbers; little if any genomic DNA remains. This method employs guanidium isothiocyanate to lyse cells and simultaneously inactivate ribonucleases rapidly. The cellular RNA is purified from the lysate via ultracentrifugation through a cesium chloride or cesium trifluoroacetate cushion since rna is more tube after 12 to 24 hours of centrifugation at 232, 000rpul? Of th dense than DNA and most proteins, it pellets at the bott This classic method yields the highest-quality RNA of any avail- able technique. Small RNAs(e. g, 5S RNA and tRNAs) cannot be prepared by this method as they will not be recovered(Mehra 1996). The original procedures were time-consuming, laborious, and required overnight centrifugation. The number and size of amples that could be processed simultaneously were limited by the number of spaces in the rotor. Commercial products have been developed to replace this lengthy centrifugation (Paladichuk, 1999)with easier, less time-consuming methods. However, if the goal were to isolate very high-quality RNA from a limited number of samples, this would be the method of choice (Glisin, Crkuenjakov and Byus, 1974) Single- and Multiple Step Guanidium Acid-Phenol Method Faster, fewer steps, prone to genomic DNA contamination, some what cumbersome if large sample numbers are to be processed The guanidium-acid phenol procedure has largely replaced the cesium cushion method because RNA can be isolated from a large number of samples in two to four hours(although somewhat cum bersome) without resorting to ultracentrifugation. RNA mole cules of all sizes are purified, and the technique can be easily scaled up or down to process different sample sizes. The single step method( Chomczynski and Sacchi, 1987) is based on the propensity of RNa molecules to remain dissolved in the aqueous phase in a solution containing 4 M guanidium thiocyanate, pH 4.0, in the presence of a phenol/chloroform organic phase. At this low pH, DNA molecules remain in the organic phase, whereas proteins and other cellular macromolecules are retained at the Martin et alinactivate RNases. The lysate is then processed in one of several ways to purify the RNA away from protein, genomic DNA, and other cellular components. A brief description of each method along with the time and effort involved, the quality of RNA obtained, and the scalability of the procedures follow. Guanidium-Cesium Chloride Method Slow, laborious procedure, but RNA is squeaky clean; unsuitable for large sample numbers; little if any genomic DNA remains. This method employs guanidium isothiocyanate to lyse cells and simultaneously inactivate ribonucleases rapidly.The cellular RNA is purified from the lysate via ultracentrifugation through a cesium chloride or cesium trifluoroacetate cushion. Since RNA is more dense than DNA and most proteins, it pellets at the bottom of the tube after 12 to 24 hours of centrifugation at ≥32,000rpm. This classic method yields the highest-quality RNA of any avail￾able technique. Small RNAs (e.g., 5S RNA and tRNAs) cannot be prepared by this method as they will not be recovered (Mehra, 1996). The original procedures were time-consuming, laborious, and required overnight centrifugation. The number and size of samples that could be processed simultaneously were limited by the number of spaces in the rotor. Commercial products have been developed to replace this lengthy centrifugation (Paladichuk, 1999) with easier, less time-consuming methods. However, if the goal were to isolate very high-quality RNA from a limited number of samples, this would be the method of choice (Glisin, Crkuenjakov and Byus, 1974). Single- and Multiple Step Guanidium Acid-Phenol Method Faster, fewer steps, prone to genomic DNA contamination, some￾what cumbersome if large sample numbers are to be processed. The guanidium-acid phenol procedure has largely replaced the cesium cushion method because RNA can be isolated from a large number of samples in two to four hours (although somewhat cum￾bersome) without resorting to ultracentrifugation. RNA mole￾cules of all sizes are purified, and the technique can be easily scaled up or down to process different sample sizes. The single￾step method (Chomczynski and Sacchi, 1987) is based on the propensity of RNA molecules to remain dissolved in the aqueous phase in a solution containing 4M guanidium thiocyanate, pH 4.0, in the presence of a phenol/chloroform organic phase. At this low pH, DNA molecules remain in the organic phase, whereas proteins and other cellular macromolecules are retained at the interphase. 204 Martin et al