[Previous] [Top] [Next] Whole organs are homogenised in
guanidinium isothiocyanate and then phenol-chloroform extracted
to remove much of the protein and DNA content of the lysate. The
partially purified lysate is then layered on to a dense caesium
chloride cushion. The buoyant density of most RNAs in caesium
chloride is much greater than that of other cellular components
(> 1.8 g/ml). During ultracentrifugation, the RNA pellets at the
bottom of the tube, the DNA bands in the caesium chloride
cushion and the protein floats in the guanidinium lysis buffer.
Small RNAs, eg 5s rRNA and tRNAs do not sediment well through
CsCl. Large amounts of very high quality total RNA in good yield
can be obtained from adult organs. It is free of protein and DNA
contamination. Intact RNA from RNase-rich tissues such as
pancreas can be consistently isolated using this method.
Although not as labour-intensive as the other methods, it is
still not suited to the preparation of large numbers of samples.
Day 1 Homogenise tissues and