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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 supernantant with a vacuum aspirator down to the last centimeter 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 aliquots 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).
(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.
3. 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.
| 250g GITC | OR 94.6g | |
| 293 ml DDW | 110ml | |
| 17.6 ml 0.75M Na citrate | 6.6ml | |
| 8.8 ml 30% sarcosyl | 3.3ml | |
| add 2ME immediately before use: 0.72 ml/100 ml | 1.44ml | |
| Total vol. 528ml | 200ml total | |
Store in light proof vessel
4. Homogenize tissue and transfer to SS34 tubes
5. Add 1ml 2M NaAc pH4, 10 ml acid phenol, 2 ml CHCl3, shake 10 sec.
Acid Phenol
Melt phenol at 60¡
Equilibrate 2x against DDW and add 0.1% hydroxyquinolone
6. Spin 12K 20' SS34
7. Collect SN and add 12 ml isopropanol, spin again at 12K for 20'
8. Resuspend pellet in in 10 ml GITC buffer. Add NaAc 1 ml, acid phenol 5ml, CHCl3 1 ml, shake 10 sec.
9. Spin at 12K 20', collect SN and add 12 ml isopropanol
10. Spin 12K 20'
11. 5 ml 70% ethanol rise
12. 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.