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

• 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