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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 assemble caesium chloride gradients
Day 2 Dissemble gradients, purify and quantitate RNA and check its integrity
Polytron or equivalent
Ultracentrifuge with swing-out rotor
Polyallomer 13 ml ultracentrifuge tubes (autoclaved if desired)
18 Store the RNA at -70oC
2 The ratio of the 260 : 280 UV absorbance readings (should be > 2.0 for clean RNA) is often poor for GIT-isolated tissues and the UV absorbance at 260 nm may bear little relationship to the amount of RNA present when checked by gel electrophoresis. Contaminating trace amounts of GIT or phenol interfere with UV absorbance by RNA at these wavelengths
A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter and H. M. Goodman (1977) Rat insulin genes : construction of plasmids containing the coding sequences Science 196:1313 -Reference #236 Tan Lab Library 07-94>
J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294-9Reference #470 Tan Lab Library 07-94>
V. Glisin, R. Crkvenjakov and C. Byus (1974) Ribonucleic acid isolated by cesium chloride centrifugation Biochemistry 13:2633-7Reference #166 Tan Lab Library 07-94>
B. E. Faulkner-Jones, D. S. Cram, J. Kun and L. C. Harrison (1993) Localisation and quantitation of expression of two glutamate decarboxylase genes in pancreatic b-cells and other peripheral tissues of mouse and rat Endocrinol 133:2962 - 2972
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1 Phenol and GIT are miscible. Chloroform must be added to searate the two phases. Heating the GIT-phenol solution to 65oC increases the efficiency of the organic solvent extraction steps 2 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 3 If the RNA yield is expected to be high, the rotor can be stopped after 16 - 18 hours 4 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 5 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH <8.0. Water saturated phenol has a pH of ~ 4.0 6 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination