This method relies on the strong chaotropic nature of the reagents involved to completely denature any ribonuclease (RNase) present in the sample. After lysis in guanidine thiocyanate buffers there are two possibilities for isolation of the RNA. One method involves a series of differential precipitation steps in guanidine hydrochloride (1). The alternative, detailed here, involves centrifugation of the samples on a cushion of 5.7M CsCl (2,3). The RNA passes through this cushion, whereas the DNA and the majority of other cellular macromolecules remain above the cushion.

This method of RNA extraction relies on a relatively gentle lysis procedure that should burst the cells, but leave the nuclei intact. Contamination of a relatively RNase-free cytoplasmic environment with nuclear nucleases is thus minimised. Next the polysomes are dissociated with SDS and proteinase K and finally the protein is removed by several phenol/choloroform extractions (1,2).

For the majority of purposes, such as restriction mapping and sizing of cloned fragments, electrophoresis of DNA in horizontal agarose gels is perfectly adequate. As the size of the fragments of interest decreases to below 1000 base pairs (bp), however, the resolution deteriorates significantly, and an alternative is required. Polyacrylamide gels from 5% acrylamide upward provide a convenient system for analysis of fragments down to 10 bp. This type of gel is also very useful for checking for efficient blunt-end ligation of linkers if the linkers are labeled with (32)P.

This article details two methods for separation and visualization of RNA under nondenaturing conditions, i.e., where the secondary structure of the molecules is left intact during electrophoresis. The first method describes electrophoresis in a 2% (w/v) agarose gel in a dilute, neutral phosphate buffer. The second deals with electrophoresis in a linear gradient of polyacrylamide based on the buffer system of Loening (1).

The separation of RNA on the basis of size by sucrose gradient fractionation is a technique frequently employed in a cloning strategy (1-3). After production of poly(A)(+) mRNA by affinity chromatography (see Chapter 16 ) the RNA can be fractionated once or twice on sucrose gradients to produce a subpopulation of mRNA enriched for a particular species. The RNA fractions from the gradient can be assayed by in vitro translation and subsequent immunoprecipitation or bioassay of the products (see Chapters 19 - Chapters 22 ).