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This method can be used for some adult tissues, most cell lines and embryos. The protocol is a modified version of that described by Auffray and Rougeon. Harvested tissues are homogenised in urea-lithium and the relatively insoluble lithium-RNA salts are precipitated overnight at 4oC. Much of the DNA remains in solution. The recovered lithium-RNA is acid phenol-chloroform extracted to remove contaminating proteins. The use of acid phenol also selectively removes DNA. Urea is a slower denaturant than guanidinium, but provided the initial tissue source is not too rich in RNase, the resulting RNA is usually largely intact, but it is always DNA contaminated. The yields from this method are good
Protocol
General
Day 1 Harvest and homogenise tissues. Precipitate overnight
Day 2 Isolate and purify RNA
Day 3 Formaldehyde-agarose gel check recovered RNA
Reagents All reagents must be made with sterile MilliQ water. Use
DEPC with care - it may inhibit subsequent enzyme reaction
Phosphate-buffered saline 6M urea, 3M lithium chloride, 0.5% SDS lysis buffer. Store at 4oC 3M sodium acetate pH 6.0 20% SDS in sterile water Sterile water 100 mM DTT RNasin RNase inhibitor (Promega) Water-saturated acid phenol Chloroform 8M LiCl
Equipment
Polytron or equivalent
Sorval centrifuge and sterile glass (Corex) tubes for recovery of RNA
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. Kill the animals by either cervical dislocation or by carbon dioxide asphyxia. Remove the tissues immediately and either Homogenise in lithium-urea lysis buffer1 with a Polytron or equivalent, or Snap-freeze in a liquid hexane bath cooled on dry ice and 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 Wash adherent cell cultures with ice cold PBS, and then completely lyse in the culture flask with 10 ml of lysis buffer / 108 to 109 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 Add SDS to a final concentration of 0.5% 4 Transfer the lysates to sterile 30 ml Corex tubes and leave at 4oC overnight
Day 2
5 Recover the RNA by centrifugation at 1 000 g for 30 minutes at 4oC2. Discard the supernatant and fully resuspend the pellet in 10 ml of 10 mM sodium acetate and 0.5% SDS 6 Extract the solution twice with acid-phenol 3 and once with acid phenol-chloroform 7 Precipitate the RNA with one volume of isopropanol and 0.1 volumes of lithium chloride 4 and recover by centrifugation at 1200g for 10 minutes 8 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 water5 9 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm
Day 3
10 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis
11 Store the RNA at -70oC
Notes
The ratio of the 260 : 280 UV absorbance readings (should be > 2.0 for clean RNA) may be poor for lithium-urea-phenol 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 phenol interfere with UV absorbance by RNA at these wavelengths
References
Reference #4 C. Auffray and F. Rougeon (1980) Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumour RNA Eur J Biochem 107:303-314
This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95. 1 Use 10 ml of lysis buffer per gram of tissue 2 Prolonged centrifugation makes the pellet very difficult to resuspend 3 RNA partitions into the aqueous phase and DNA partitions into the phenolic phase when the pH <8.0. Water saturated phenol has a pH of ~ 4.0 4 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 5 The pellet may be resuspended in lithium-urea, and the phenol-chloroform extractions repeated a second or even third time. This is necessary for pancreas