MRNA extraction

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Homogenize TRIZOL (10 mg ----- 200 uL of trizol) Incubate for 5 mts at room temperature


Add chloroform (40 uL for 200 uL of tizol) Mix for 15 secs Incubate for 5 mts at room temperature Centrifuge 20 mts at 12000 (11.3) for 20 mts at 4 degrees Aqueous phase transfer to RNase free tube


Add ice cold ISOPROPANOL (100 uL for 200 uL of trizol) Mix for 15 secs Incubate for 30 mts or overnight at -20 degrees Centrifugation at 12000 (11.3) for 20 mts at 4 degrees Supernatant removed


Add ice cold 75 % EtOH (200uL for 200 uL of trizol) Pellet unstuck from the bottom/border Centrifuge at 7500 (8.9) for 10 mts at 4 degrees Remove EtOh

REPEAT x 2



Add 0.1 volume of 10xx turbo DNase buffer Add 1 uL turbo DNase Incubation for 20 – 45 mts at 37 degrees (30 mts) Add 0.1 volume of DNase inactivation reagent 2 mts incubation at room temperature Centrifuge at 10000 (10.3) for 1.5 mts (2 mts) at 4 degrees Transfer supernatant to new tubes Measure concentration