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RESEARCH DIVISION Laboratory Manual

 


 

Acid Phenol Method for Isolation of RNA from Adult Mouse Tissues

(modified from Chomczynski, Analytical Biochemistry 162: 156)

  1. Kill mouse, dissect tissues and freeze on dry ice in 50 ml tube
  2. Grind in mortar and pestle under liquid N2.

Guide to amounts of mouse tissues:

- 0.5 liver
- 4 kidneys
- 8 testes
- 0.75g pectoral and quad. muscles
- 4 hearts
- 4 submandibular salivary glands
- 4 pairs of lungs
- 4 spleens
- section of sm. intestine

Do brains with proteinase K method.

  1. Add powdered tissue to 10 ml GITC buffer. NOTE: if you add tissue to a capped tube be careful of the high pressure as liquid N2 in sample turns to a gas. It can explode the tube.
GITC buffer

4 M GITC, 25 mM Na citrate pH7, 0.5% sarcosyl, 0.1M 2ME (Store in light proof vessel)

GITC 250g 94.6g
DDW 293ml 110ml
0.75M Na citrate 17.6ml 6.6ml
30% sarcosyl 8.8ml 3.3ml
Total Volume 528ml 200 ml
add 2ME immediately before use: 0.72 ml/100 ml 1.44ml/ 200 ml
  1. Homogenize tissue and transfer to SS34 tubes
  2. Add 1ml 2M NaAc pH4, 10 ml acid phenol, 2 ml CHCl3, shake 10 sec.
Acid Phenol
- Melt phenol at 60C
- Equilibrate 2x against DDW and add 0.1% hydroxyquinolone
  1. Spin 12K 20' SS34
  2. Collect SN and add 12 ml isopropanol, spin again at 12K for 20'
  3. Resuspend pellet in in 10 ml GITC buffer. Add NaAc 1 ml, acid phenol 5ml, CHCl3 1 ml, shake 10 sec.
  4. Spin at 12K 20', collect SN and add 12 ml isopropanol
  5. Spin 12K 20'
  6. 5 ml 70% ethanol rise
  7. Resuspend in 5 ml STE/0.5M NaCl/Proteinase K 200 g/ml and proceed with polyA selection (as in manual below).

This method is the most effective we have used for preparing RNA from adult mouse tissues.

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