1. Re-suspend the homogenized, cultured cells in 1ml of 1M TRI Reagent. If the cells are grown in monolayer, add 1ml of 1M TRI reagent to the wells. In case the cells are grown in suspension, centrifuge and add 1ml of 1M TRI Reagent to the pellet. The TRI reagent is used to lyse the cells.
2. Incubate the samples for 5 minutes at room temperature (RT).
3. After incubation, transfer the samples to 1.5ml eppendorf tubes.
4. Add 100μl of chloroform to the samples and shake the tubes for around 15 seconds to mix thoroughly and incubate the samples for 5-15 minutes at RT.
5. Centrifuge the samples at 12000 x g for 8 minutes at 4°C. This will form 3 layers in the eppendorf tube. The top, clear aqueous layer contains the total RNA, followed by a layer of interphase DNA and finally, a layer of protein and lipids.
6. Carefully pipette the top aqueous layer that contains the RNA into new eppendorf tubes.
7. To these samples, add 500μl of isopropanal and mix thoroughly, vortexing for 5-10 seconds. Incubate the samples for 5-10 minutes at RT.
8. Centrifuge the samples at 12000 x g for 8 minutes at 4-25°C and discard the supernatant.
9. Wash the pellet with 1ml of 75% ethanol in the eppendorf tubes. Mix thoroughly by vortexing.
10. Centrifuge the sample at 7500 x g for 5 minutes. Discard the ethanol. This will leave behind a white RNA pellet. Airs dry the RNA pellet briefly.
11. Re-suspend the pellet in approximately 80μl of DEPC water. The volume of DEPC water added to dissolve the pellet is dependant on the size of pellet. If the pellet size appears big, increase the volume of DEPC water. If the pellet size appears small, decrease the volume of DEPC water according.
12. Incubate the mixture at 55-60°C for approximately 10 minutes to completely dissolve the RNA pellet
13. The RNA concentration can now be determined using Nanodrop.
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