Homogenize 50 to 100 milligrams (mg) of tissue in 1 milliliter (ml) of TRIzol reagent with a glass-Teflon or power homogenizer to release the RNA. The method will vary with cells grown in monolayer or in suspension; in these cases cell lysis can be accomplished through pipetting several times instead.
Isolate excess proteins, fat, polysaccharides and plant tissues with the following optional centrifugation step: Spin your tubes in the centrifuge at a setting of no more than 12,000 rcf (relative centrifugal force) for 10 minutes at a temperature of 2 to 8 degrees Celsius (C). Remove any top fat layer and discard. The resulting supernatant will hold the RNA; remove this to a fresh tube and discard the pellet.
Incubate the homogenized sample for five to 10 minutes at room temperature (15 to 30 degrees Celsius). Then add 0.2 ml of chloroform per 1 ml of TRIzol. Cap the tube securely and shake by hand for 15 seconds. Incubate again --- for two to three minutes at room temperature --- and then centrifuge at no more than 12,000 rcf for 15 minutes at 2 to 8 degrees C. The mixture should now be in three phases: a lower red, phenol-chloroform phase; an interphase; and a colorless upper phase called the aqueous phase. Remove the aqueous phase to a fresh tube --- this phase holds the RNA.
Add 0.5 ml of isopropyl alcohol per 1 ml TRIzol to the aqueous phase and then incubate for 10 minutes at 15 to 30 degrees C. Follow the incubation with centrifugation at no more than 12,000 rcf for 10 minutes at 2 to 8 degrees C. You should be able to see a gel-like RNA pellet at the bottom of the tube.
Discard the supernatant by carefully pipetting off. Air-dry the RNA pellet for five to 10 minutes but don't dry it completely or you won't be able to resuspend it. Dissolve the pellet in RNase-free water or 100 percent deionized formamide by mixing with a pipette and then incubating for 10 minutes at 55 to 60 degrees C.
Store at -70 degrees C.