RNA ISOLATION FROM WHOLE BLOOD

HOMOGENIZATION Dilute 1 ml blood with 1 ml water. (to dilute the high levels of contaminating material in blood) Add 6 ml Trizol. (maintains integrity of RNA while disrupting and dissolving cells) Vortex and pass through a 21G needle. (to lyse the cells) PHASE SEPARATION Incubate homogenized samples for 5 minutes at 15 to 30oC. (dissociation of nucleoprotein complexes) Add 1.6 ml of chloroform and cap tube. (dissolves organic substances) (use 0.2 ml chloroform/0.75 ml Trizol) Shake the tube by hand for 15 seconds and incubate at c Centrifuge 12,000 x g for 15 minutes at 2 to 8oC. Mixture now has three layers: lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase containing the RNA. Volume of aqueous phase is about 70% of the volume of Trizol used for homogenization. RNA PRECIPITATION Transfer the aqueous phase to a clean tube. Save the organic layer if DNA isolation is desired. Add 4 ml isopropyl alcohol. (0.5 ml isopropyl alcohol/0.75 ml Trizol) Incubate at 15 to 30oC for 10 minutes. Centrifuge 12,000 x g for 10 minutes at 2 to 8oC. (RNA precipitate forms a gel-like pellet on the side and bottom of the tube) RNA WASH Remove the supernatant. Wash the RNA pellet once with 8 ml 75% Ethanol (1 ml ethanol/0.75 ml Trizol) Mix the sample by vortexing. Centrifuge 7500 x g for 5 minutes at 2 to 8oC. REDISSOLVING THE RNA Briefly air dry the RNA pellet. Do not let the pellet dry completely. Dissolve the RNA in RNase-free water and incubate for 10 minutes at 55 to 60oC.