PROCEDURE FOR RNA ISOLATION

• Grind 100 mg of tissue with liquid nitrogen in a sterile pestle mortar. Alternatively, tissue can be homogenized using a glass-Teflon or Polytron homogenizer or another suitable homogenizer
• Add 1 ml of Tri-RNA Reagent to 100 mg of tissue
• Incubate the homogenate for 5 minutes at room temperature with intermittent mixing on a vortex mixture
• Add 0.2 ml of molecular biology grade chloroform (not provided) and mix vigorously
• Centrifuge at 12,000 -14,000 x g for 10 minutes at 4°C to separate the phases
• Transfer the RNA present in the upper aqueous phase to a fresh 1.5 ml microfuge tube*
• Precipitate RNA by adding an equal amount of isopropanol (IPA), and mix properly by inverting tubes followed by incubation at room temperature for 10 minutes#
• Recover the precipitated RNA by centrifugation at 15,000 x g for 15 minutes at 4°C
• Discard the supernatant, and wash the RNA pellet with 0.5 ml of ice-cold 75% ethanol. Centrifuge at 12,000 -14,000 x g for 5 minutes at 4°C, and carefully discard the supernatant
• Recentrifuge briefly and carefully remove any residual supernatant without disturbing the RNA pellet
• Allow the RNA pellet to vacuum or air dry at room temperature for 10-15 minutes
• Resuspend the RNA in DEPC-treated water or Tris-EDTA, pH 8.0
• Store the RNA sample at -80 °C