Total RNA Isolation using the TRIzol Reagent

NOTE: This protocol is for cells growing on a monolayer.

1. Remove media from the plate using suction. Immediately lyse cells directly on the plate by adding 5 ml TRIzol reagent in each 10-cm plate and pipetting cells up and down several times. (The amounts of reagent for other plate sizes are: 1 ml/3.5 cm plate, 3 ml/6 cm plate, 11 ml/15 cm plate). Transfer lysates in orange cap tubes.

2. Incubate samples for 5 min at room temperature. Then add 1 ml of chloroform (or 0.2 ml/1 ml of TRIzol reagent used). Close tubes securely (BE CAREFUL, THE REAGENT CONTAINS PHENOL). Shake tubes by hand for approx. 15 seconds and incubate at room temperature for 2-3 minutes.

3. Centrifuge samples at 4,000 rpm at 4^ο C for 30 min using the Beckman GS-6KR floor centrifuge.

4. Following centrifugation the sample will separate into a lower red phase, an interface and a colorless upper phase. Transfer the clear upper phase (approximately 3 ml) in a clean marked orange cap tube.

5. Precipitate the RNA by adding 2.5 ml of isopropanol in each tube. Mix by inverting the tubes, incubate for at least 10 minutes at room temperature and recover RNA by centrifugation at 4,000 rpm at 4^ο C for 30 min using the Beckman GS-6KR (if you want you can stop here, the RNA can be stored at -20^ο C under isopropanol).

6. Remove supernate and wash the RNA pellet twice with 7 ml 70% ethanol. Recover RNA after each wash by centrifugation for 10 min at 4^ο C, as described above (step 5).

7. Remove the last ethanol wash and dry the RNA pellets under vacuum for 5-10 min. Do not overdry the RNA because you are going to have hard time redissolving it. In each tube add 100 μl RNAse-free, DEPC-treated and autoclaved water. Pipette up and down several times using autoclaved Pasteur pipettes and then incubate the RNA samples for 10 minutes at 65^ο C.

8. Determine the RNA concentration by adding 5 μl from each sample in 500 μl water and measuring the absorbance at 260 nm (when absorbance is 1, the RNA concentration is 40 ng/ml). The absorbance at 280 nm should also be measured and the 260/280 ratio should be at least 1.7 (normally it is 1.85-2.0 for good quality RNA). You should expect to get at least 100 μg of RNA from each plate of Vero cells (90% confluent).

NOTE: The best way to store RNA is under 70% ethanol at -20^ο C. However, for short periods of time it can be stored in RNAse-free, DEPC-treated and autoclaved water at -80^ο C.