Ribonucleic acid extraction from samples using TRIzol Reagent

Health and Safety and Other Risks:

• Disposable gloves and a laboratory coat should be worn to avoid contact with skin and clothes when using TRIzol Reagent as it contains phenol. Tubes containing waste TRIzol Reagent must be placed into a suitable container for appropriate disposal.

• RNA extraction must be carried out in a location separate from areas where reverse transcription and PCR amplification are carried out to avoid contamination of stock reagents.

• Disposable gloves must be worn throughout the technique as RNases from the skin have an adverse affect on RNA. Precipitated extracted RNA pellets may be very small or invisible; care must be taken to avoid accidental discard. Extracted RNA should be stored at -90 to -50oC in a monitored freezer.

Health and Safety:

This protocol should be used in accordance with the current Health and Safety Policy and other relevant instructions as issued by the Institute for Animal Health. Due consideration must be given to National standards and regulations.

• Ensure any samples frozen and previously stored in TRIzol are thawed. Incubate for 5 minutes at approximately 22°C (room temperature).
• Label a fresh 2 ml skirted Sarstedt tube corresponding to each test sample to be processed; add 200 ?l of chloroform to each 2 ml tube followed by 1.0 ml of the TRIzol solution containing the sample.
• Vortex mix each tube for 10-15 seconds.
• Centrifuge the tubes for 15 minutes at 13,000 rpm at 2-8°C.
• Label a fresh 1.5 ml conical tube for each test sample and add 1.0 ?l of glycogen to each.
• Remove 500 ?l of the top phase (clear phase) from the centrifuged tubes (from stage 5.4) and add to the corresponding glycogen-containing tubes. Add 500 ?l of isopropyl alcohol to each tube.
• Vortex mix each tube for a few seconds.
• Incubate the tubes at approximately 4°C (on ice) for 10 minutes.
• Orientate the tubes in the centrifuge using the tube label so that the RNA pellet will be in an expected position. Centrifuge the tubes for 10 minutes at 13,000 rpm at 2-8°C.
• Discard the supernatant from the tubes and then add 1.0 ml of 70% ethanol to each one.
• Vortex mix the tubes for a few seconds.
• Orientate the tubes in the centrifuge as in stage 5.9 and centrifuge for 10 minutes at 13,000 rpm at 2-8°C.
• Remove as much supernatant as possible using gentle vacuum suction (or pipetting) without disturbing the RNA pellet. Air dry each tube for 2-3 minutes at approximately 22°C. Resolubilise each RNA pellet by adding 20 ?l of nuclease-free water to each tube and gentle mixing.
• Maintain the RNA samples on ice and immediately proceed with RT-PCR or store them at -90°C to -50°C until required.

Results will only become known after analysis of the subsequent reverse transcription and PCR amplification procedures.

If pipettes subsequently fail calibration checks but final results are satisfactory according to the controls, the Line Manager or Operator may still deem the results acceptable