How to isolate RNA from tissue or cells

Put on lab coat, gloves, and safety glasses. Prepare your work area and gather all necessary materials including the TRIzol Plus RNA purification kit, wash buffers, and equipment.

Add 1 mL of TRIzol reagent to 50-100 micrograms of fresh or frozen tissue or cells. Homogenize thoroughly using a tissue rotor stator homogenizer and let stand at room temperature for 5 minutes.

Add 2 mL of chloroform and shake the tube vigorously for 15 seconds. Let sit at room temperature for 2-3 minutes, then centrifuge at 12,000g for 15 minutes at 4°C to separate into three distinct layers.

Transfer 400 microliters of the colorless upper aqueous layer containing RNA to a fresh RNase-free tube. Add an equal volume of 70% ethanol and mix thoroughly by vortexing.

Transfer 700 microliters of the ethanol-RNA sample to a PureLink silicon membrane column inserted into a 2 mL collection tube. Centrifuge at 12,000g for 15 seconds and discard the flow-through.

Add 700 microliters of Wash Buffer 1 and centrifuge at 12,000g for 15 seconds. Repeat with 500 microliters of Wash Buffer 2, then dry the column by centrifuging at 12,000g for 1 minute, discarding flow-through after each step.

Insert the column into a recovery tube and add the appropriate volume of elution buffer based on starting amount. Incubate at room temperature for 1 minute, then centrifuge at 12,000g for 2 minutes to collect the purified RNA.

The ultra-pure RNA is now in the recovery tube and ready for use or storage at -20°C for future applications.