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RNA Purification from Tissue Samples

  • Homogenize tissues in 1ml TRIzol per 50-100mg tissue (use green-capped tubes)
    • Use homogenizing machine in Genomics Core (please see us for training)
    • Keep samples on ice prior to adding TRIzol reagent
  • Centrifuge sample at 13000 x g for 10 minutes at 4°C
  • Remove supernatant and place in fresh RNase-free tube (KEEP ON ICE)
  • Incubate homogenized sample/supernatant 5 minutes at room temperature to allow complete dissociation of nucleoprotein complexes
  • Add 200µl chloroform per 1ml TRIzol reagent (1:5). Shake tube vigorously by hand for 15 seconds (DO NOT VORTEX)
  • Incubate at room temperature for 3-5 minutes
  • Centrifuge sample at 13000 x g for 15 minutes at 4°C
    • Three phases will appear:
      • Top: Aqueous, colorless (RNA)
      • Middle: Intermediate
      • Bottom: Red (Phenol-Chloroform)
  • Transfer ~400µl colorless, upper phase containing the RNA to a fresh RNase-free tube
Go to kit:
  • Add one volume of 70% EtOH (made with autoclaved or RNase-free water)
  • Vortex to mix thoroughly
  • Transfer up to 700µl sample to spin cartridge
  • Centrifuge at 12000 x g for 15 seconds at room temperature. Discard flow-through
  • Continue until all sample has passed through spin cartridge
  • Add 700µl Wash Buffer I to spin cartridge
  • Centrifuge at 12000 x g for 15 seconds. Transfer spin column to a new collection tube
  • Add 500µl Wash Buffer II to spin cartridge
  • Centrifuge at 12000 x g for 15 seconds. Discard flow-through
  • Repeat previous steps with Wash Buffer II
  • Centrifuge spin cartridge at 12000 x g for 1-2 minutes to dry membrane bound with RNA. Discard collection tube and insert the spin cartridge into a recovery tube
  • Add 25-100µl RNase-free water to center of spin cartridge (dependent on original tissue size)
  • Incubate at room temperature for 1 minute
  • Centrifuge spin cartridge for 2 minutes at greater than 12000 x g at room temperature to elute the RNA from the membrane
  • Store purified RNA at -80°C or store on ice for concentration determination