David Bowtell

RNA ISOLATION USING GUANIDINE HYDROCHLORIDE

RNA isolation ; guanidine hydrochloride;
1) For adult flies 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. Some of us have found that the addition of liquid nitrogen is unnecessary.

2) Add the powder to room temp 6M GHCl, 0.1M NaOAc or 6M guanidine isothiocyanate, 0.1M NaOAc. I generally use 10-20ml per scintillation vial of flies. 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. A beaker with the guanidine stirring in it is probably a safer alternative.

3) Shear the lysate through an 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 has a lot of cuticle 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 16-18hr at 20° C.

5) Remove supernatant with a vacuum aspirator down to the last centimeter of liquid. Often a prominant band is visible ~0.5cm above the pellet. This is (?) tRNA. The pellet is usually not obvious until all the liquid is removed. Cut the tube off with a scalpel blade above the liquid and invert the tube. The pellet should be clear and gelatinous. Resuspend in ~200µl 10mM EDTA by shunting up and down with a p200. It will take several minutes. Be careful not to let the suspension touch the end of the pipette. Remove 5-10µl into 400µl 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, 200µg/ml proteinase K and 0.1% SDS (it is not necessary to phenol extract) and either oligo-dT select batchwise or over a column.

7) This works fine for adult flies, embryos, discs and probably all other stages.