Subcellular fractionation and RNA extraction

Subcellular fractionation
1. Harvest cell pellet and rinse with 1x PBS twice.
2. Resuspend the cell pellet in hypothonic buffer (10 mM Tris (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.075% NP-40, and 2 mM Ribonucleoside vanadyl complexes) gently and incubate on ice for 5 min. *Volume of hypotonic buffer is depend on the amount of cells.
3. Check membrane lysis with trypan blue. Proceed with centrifugation at 500 x g for 10 min at 4degree if >90% of cells are lysed. If not, increase the incubation time. Do not overlyse the cells.
4. Collect the supernatant (cytoplasmic fraction) and wash the pellet (nuclear fraction) with hypotonic buffer for 3 x.
5. Add 1 mL of RNA precipitation solution (RPS; 0.15 M sodium acetate (pH 5.5) in ethanol) to the cytoplasmic fraction and incubated at -20degree for at least 1 hour.
6. Vortex the Cytoplasmic fraction in RPS and centrifuge at 18000 x g for 15 min at 4degree.
7. Discard the supernatant and rinse the pellet with 70% (v/v) ice-cold ethanol.
8. Add 1 mL of Trizol to the semi-dry nuclear and cytoplasmic pellets followed by the addition of 10 microlitre of 0.5 M EDTA.
9. Heat both fractions at 65degree until the pellet dissolved with vortexing.
10. Cool the samples to room temperature and add 200 microlitre of chloroform:isoamyl alcohol (1:24).
11. Vortex the samples and centrifuge at 18000 x g at room temperature for 10 min.
12. Aliquote the aqueous supernatant into a clean microcentrifuge tube.
13. Add equal volume of isopropanol and incubate at -20degree for at least 1 hour.
14. Vortex the samples and centrifuge at 18000 x g at room temperature for 15 min.
15. Wash the pellet with 70% (v/v) ethanol.
16. Dissolve the air-dried RNA pellet in RNase-free water.