With the exception of step 7, the protocol for isolating RNA is identical to that described in the Trizol® protocol.
3. Remove cells from the -80°C freezer and place the tubes on dry ice to prevent thawing.
4. Remove one sample from the dry ice and add 1 ml of Trizol®. It is not necessary to thaw the cells prior to adding the Trizol®.
5. Lyse the cells using a L-1000 pipettor by repeated pipetting until the solution is homogeneous. Incubate at room temperature for 5 min.
6. Add 200 yl of chloroform, shake by hand 15 times and incubate at room temperature for 3 min.
7. Transfer the mixture to a 2 ml Eppendorf phase lock tube. The phase lock tube will prevent mixing of the organic and aqueous layers.
8. Centrifuge the phase lock tube in the cold for 15 min at 12,000 x G. Remove the supernatant and place into a 1.5 ml colored, microcentrifuge tubes. Colored tubes will enhance visualizing the RNA pellet.
9. Add 500 ^l of isopropanol and precipitate the RNA for 10 min at room temperature. This is a good stopping point. If necessary, the samples may be placed in the -20°C freezer overnight.
10. Place the tubes in a microcentrifuge in the cold (e.g. cold room, refrigerated microcentrifuge or microcentrifuge placed in a refrigerated unit). Orient the caps to the outside of the centrifuge's rotor. Centrifuge for 10 min at 12,000 x G. The RNA pellet should be visible at the bottom of the tube on the side that was oriented to the rotor's outside.
11. Decant the supernatant into a 2 ml microcentrifuge tube. It is not necessary to remove all of the supernatant. Add 1,000 ^l of 75% ethanol. Centrifuge for 5 min at 7,500 x g in the cold.
12. Decant the supernatant into the same 2 ml microcentrifuge tube used for the isopropanol. Briefly spin the tube containing the RNA for several seconds to bring the residual ethanol to the bottom of the tube.
13. Using a pipette, remove most of the residual ethanol, being careful not to disturb the pellet. To remove the remaining ethanol, place the tubes into a dessicator containing a porcelain platform filled with Drierite. Connect to house vacuum for 5 min. It is not necessary to completely dry the sample since the residual water will not affect the reverse transcription step.
14. Dissolve the RNA pellet in 30-50 ^l of molecular biology grade water and place the tubes on ice. RNA may be stored at -80° C, however it is recommended that RNA be converted to cDNA on the same day of the isolation.
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