m6bii

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

Mini-ultracentrifuge method

Cultured cells, small pieces of tissue or embryos are homogenised in guanidinium isothiocyanate and the lysate is 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.

The total RNA obtained is of very high quality, in good yield and is pure from protein and DNA contamination as long as the capacity of the gradient is not exceeded. 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.

Phosphate-buffered saline 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 2.2 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. Tissues Only small pieces of tissue or embryos can be used : whole adult organs will exceed the capacity of the gradients unless phenol : chloroform extracted first. Remove tissues or embryos immediately and either - homogenised in 5 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 Cells Wash adherent cell cultures with ice cold PBS, and then completely lyse in the culture flask with 5 ml GIT lysis buffer / 106 to 107 cells. Draw the lysate several times through an 18 gauge needle with a 20 ml syringe to shear genomic DNA. The lysate may also be sheared using the Polytron 3 Precipitate crude nucleic acids with 1 volume of isopropanol and 1 / 10 volumes of 8M LiCl and recover by centrifugation 4 Resuspended in 1 ml of 4M GIT lysis buffer. Remove insoluble material from the cell and tissue lysates by centrifugation. 5 Make a 600 ml 5.7M CsCl cushion in the bottom of each ultracentrifuge tube - remove all bubbles with a sterile pipette tip 4 Add 100 ml of 5.7M CsCl, 1 mM EDTA, 30 ml of 10 M b-mercaptoethanol and 30 ml of 20% (w/v) N-lauryl sarcosine to each GIT-tissue lysate 1. Mix and then layer CsCl-GIT lysate over the 600 ml CsCl cushion. 5 Balance ultracentrifuge tubes to within 0.05 g and centrifuge at 45 000 rpm (180 000 g at the tube bottom in the TLS-55 rotor in a Beckman TL-100 ultracentrifuge) for 6 - 8 hours 2 at 20oC 3.

'Day 2'

6 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 7 Resuspend the RNA pellet in 100 ml of 4M GIT, 25 mM Na Acetate pH 6.0 by gently aspirating up and down with a wide bore pipette. 8 Extract the resuspended RNA with and equal volume of acid-phenol 4 until the interface is clear 9 Extract once with chloroform 10 Precipitate with 1 volume of isopropanol and 1 / 10 volume of 8M LiCl 5 and recover by centrifugation at 1200g for 10 minutes 11 Wash the pellet in 70% ethanol and then resuspend in 20 - 100 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water. 12 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm. 13 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis

14 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

2 After resuspending the RNA pellet in GIT - steps 4 or 7 - the solution may be frozen at -70oC prior to further purification

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 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 2 If the RNA yield is expected to be high, the rotor can be stopped after 6 hours 3 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 4 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 5 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 (4) and (5) reduce the amount of contamination