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RESEARCH DIVISION Laboratory Manual

 
 

RNA Isolation Using Guanidinium Hydrochloride

  1. For tissues that are frozen, first grind to a fine powder in a mortar and pestle under liquid nitrogen. Use a large pestle to prevent "blasting" the powder out when the nitrogen is added.
  2. Add the powder to room temp 6M GHCl, 0.1M NaOAc pH5.5. I generally use 10-20ml per gram of tissue. It is better to add too much than too little liquid but first consider the number of samples you are spinning in step 4. (A SW41 ;can take about 42ml of GHCl lysate per run). NOTE: if you add the powder to a 50ml tube be careful shaking it up once the lid is screwed on. Release of the dissolved nitrogen can blow the lid off. I suggest you wear glasses at all the stages involving handling of the GHCL.
  3. Shear the lysate thru a 18ga. needle and then preclear twice at 10-15K in the SS34. It is important to get rid of all the particulate material. The first preclear often has a lot of debris floating on the top.
  4. Pretreat polyallomer tubes with 0.1M NaOH and rinse with DEPC DDW. Add 5.0ml of 5.2M CsCl, 10mM EDTA and then layer the supernatant over the top. Spin (SW41, SW40) 30K 18 hr+ at 20°C.
  5. Remove supernatant with a vacuum aspirator down to the last centimetre of liquid. Cut the tube off with a scalpel blade above the liquid and invert the tube. The pellet should be clear and gelatinous. Resuspend in ~200ul of 10mM EDTA by shunting up and down with a p200. It takes several minutes. Be careful not to let the suspension touch the end of the pipette. Remove 5-10ul into 400ul of 10mM EDTA for OD. Add the remainder to ethanol/NaCl. It is best to store the RNA as a fine precipitate in ethanol at -70°C from which aliquot’s can be removed.
  6. To oligo-dT select, spin down RNA (very briefly if you have a lot otherwise the pellet can be hard to resuspend). Resuspend in 10mM Tris, 1mM EDTA, 0.5M NaCl, 200ul/ml proteinase K and 0.1% SDS (it is not necessary to phenol extract) and either oligo-dT select batchwise or over a column (see method below).
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998