m6biii

Isolation of total cellular RNA using 4M guanidinium isothiocyanate lysis buffer and caesium chloride

ultracentrifugation : Large scale isolation

of total cellular RNA from adult organs

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.

Protocol

General

Day 1 Homogenise tissues and assemble caesium chloride gradients

Day 2 Dissemble gradients, purify and quantitate RNA and check its integrity

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reactions.

Guanidinium lysis buffer :- 4M Guanidinium isothiocyanate, 25 mM sodium acetate pH 6.0 and 1 mM EDTA pH 8.0. Store at room temperature - it will keep for months but it is light sensitive 5.7M caesium chloride, 25 mM acetate pH 6.0, 1 mM EDTA, 0.1% DEPC. Autoclave and store at room temperature 20% (w/v) N-lauryl sarcosine 10 M b-mercaptoethanol Sterile water 100 mM DTT RNasin RNase inhibitor (Promega) Water-saturated acid phenol Chloroform 8M LiCl

Equipment

Polytron or equivalent

Ultracentrifuge with swing-out rotor

Polyallomer 13 ml ultracentrifuge tubes (autoclaved if desired)

Methods

In advance

1 Clean Polytron probe with 3 changes of distilled water. If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA

Day 1

2 Harvest tissues or cells for RNA isolation. Killed animals by either cervical dislocation or by carbon dioxide asphyxia. Remove tissues immediately and either - homogenise in up to 25 ml of GIT lysis buffer with a Polytron or equivalent, or - snap-freeze in a liquid hexane bath cooled on dry ice. Store snap-frozen tissues at -70oC until required for RNA isolation. NB do not store too long, especially if tissue is RNase rich eg pancreas 3 Add 0.5 volumes of acid-phenol, vortex, and then phase-separate by the addition of 0.5 volumes of chloroform 1. Vortex and recover aqueous phase by centrifugation. 4 Repeat the phenol-chloroform extractions until the interface is clear. 5 Precipitate crude nucleic acids with 1 volume of isopropanol and 1 / 10 volume of 8M LiCl. Recover by centrifugation. 6 Wash the pellet in 70% ethanol and then resuspend in 7 ml of 4M GIT 7 Make a 4 ml 5.7M CsCl cushion in the bottom of each polyallomer ultracentrifuge tube - remove all bubbles with a sterile pipette tip 8 Add 1 ml of 5.7 M CsCl, 1 mM EDTA, 4 x 30 ml drops of 10 M b-mercaptoethanol and 8 x 30 ml drops of 20% (w/v) N-lauryl sarcosine to each GIT-tissue lysate 2. Mix and then layer CsCl-GIT lysate over the CsCl cushion. 9 Balance ultracentrifuge tubes to within 0.05 g and centrifuge at 33 000 rpm (180 000 g at the tube bottom in the SW40Ti rotor) for 21 hours 3 at 20oC 4

Day 2

10 Remove the GIT-CsCl by placing a pipette at the air-liquid interface and gently aspirating the tube contents. Swab the inside of the tube to remove residual GIT-CsCl, taking care not to touch the clear, gelatinous RNA pellet at the tube bottom 11 Resuspend the RNA pellet in 400 - 750 ml of 4M GIT, 25 mM Na Acetate pH 6.0 by gently aspirating up and down with a wide bore pipette. 12 Extract the resuspended RNA with and equal volume of acid-phenol 5 until the interface is clear 13 Extract once with chloroform 14 Precipitate with 1 volume of isopropanol and 1 / 10 volume of 8M LiCl 6 and recover by centrifugation at 1200g for 10 minutes 15 Wash the pellet in 70% ethanol and then resuspend in 50 - 500 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water. 16 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm. 17 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis

18 Store the RNA at -70oC

Notes

1 This method should give intact, pure total RNA. Both 28 and 18 s rRNA bands are clearly seen. The 5 s rRNA can be seen only if CsCl is added to the GIT-tissue lysate

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

References

Reference #143 Tan Lab Library 07-94> Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular Cloning, A Laboratory Manual, Second Edition. Cold Spring Harbour Laboratory Press. Reference #151 Tan Lab Library 07-94> Davis LG, Dibner MD, Battey JF. 1986 Basic Methods in Molecular Biology. Elsevier. New York Reference #237 Tan Lab Library 07-94>

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 18:5294-9

Reference #470 Tan Lab Library 07-94>

V. Glisin, R. Crkvenjakov and C. Byus (1974) Ribonucleic acid isolated by cesium chloride centrifugation Biochemistry 13:2633-7

Reference #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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This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95.

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