Preparation of RNA
Procedure
Prepare the cell sample from which RNA to be isolated
For monolayer cells:
1. Rinse cell monolayer twice with ice-cold PBS, and the cells are scraped to a beaker containing ice cold PBS and transfer the mixture to a centrifuge tube
2. Centrifuge the solution at maximum speed for 15 sec in a microcentrifuge maintaining temperature at 4°C and collect the cells.
For Cell Grown in Suspension
1. Harvests the cell by centrifugation in a table top centrifuge for 5-10 minutes at room temperature
2. Remove the medium and resuspend the cells in one-half original culture volume ice-cold PBS. Repeat the process
The Process
1. Transfer the cell to micro centrifuge tube containing 400 µl ice cold Lysis buffer and place it on ice for 5 minutes.
2. Centrifuge the mixture in a microcentrifuge for 2 minutes at maximum speed.
3. Collect the supernatant in a clean tube and add 4 µl of 20% SDS to denature the protein part. Mix immediately by vortexing.
4. To digest the protein part, add 2.5 µl of 20 mg/ml proteinase K. Incubate 15 min at 37°C.
5. Add 400 µl of 25:24:1 phenol/chloroform/isoamyl alcohol, room temperature., mix thoroughly and collect in a centrifuge tube
6. The resultant mixture is centrifuged for 3 minutes in a microcentrifuge at maximum speed .Carefully remove the supernatant to a clean tube and repeat the step 5
7. Collect the aqueous upper phase to a clean tube and add 400 μl of 24:1 chloroform/isoamyl alcohol. Mix thoruogly by vortexing 15 to 30 sec
8. Centrifuge the mixture for 1 minute in a microcentrifuge at maximum speed, in room temperature and collect the upper phase into a clean tube
9. Add 1 ml of 95% ethanol and 40 µl of 3 M sodium acetate (DEPC-treated) to the RNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes. Store the sample at -20°C overnight .
10. Centrifuge at maximum speed in a microcentrifuge for 15 min at 4°C, decant the supernatant, and drain inverted on a paper towel
11. Add 1 ml 80% ethanol /25% 0.1 M sodium acetate to the pellet and rinse the mixture.
12. Store at -20°C until needed.
