TRIZOL RNA Isolation Protocol

Protocol

1. Homogenize tissue samples in 1 ml of TRIZOL reagent per 50 to 100 mg of tissue using a glass-Teflon or power homogenizer (e.g. Polytron, Tekmar’s TISSUEMIZER). The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for the homogenization.

2. Rinse cell monolayer with ice cold PBS once. Lyse cells directly in a culture dish by adding 1 ml of TRIZOL Reagent per 3.5 cm diameter dish and scraping with cellscraper. Pass the cell lysate several times through a pipette. Vortex thoroughly. The amount of TRIZOL reagent added is based on the area of the culture dish (1 ml per 10 cm2) and not on the number of cells present. An insufficient amount of TRIZOL Reagent may result in DNA contamination of the isolated RNA.

3. Spin cells for 5 min at 300 X g. Remove media and resuspend cells in ice cold PBS. Pellet cells by spinning at 300 X g for 5 min. Lyse cells with TRIZOL Reagent by repetitive pipetting or by passing through syringe and needle. Use 1 ml of the reagent per 5-10 X 106 of animal cells.

4. Incubate the homogenized sample for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Centrifuge to remove cell debris. Transfer the supernatant to new tube.

5. Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely. Vortex samples vigorously for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 2 to 80C. Following centrifugation, the mixture separates into lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube. Measure the volume of the aqueous phase (The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization).

6. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000x g for 10 minutes at 2 to 4oC. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.

7. Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial homogenization. Mix the samples by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at 2 to 8 oC. Repeat above washing procedure once. Remove all leftover ethanol.

8. Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by centrifuge under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/A280 ratio < 1.6. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip.

9. Dilute 1 μl of RNA with 39 μl of DEPC-treated water (1:40 dilution). Using 10 μl microcuvette, take OD at 260 nm and 280 nm to determine sample concentration and purity. The A260/A280 ratio should be above 1.6. Apply the convention that 1 OD at 260 equals 40 µg /ml RNA.

REFERENCE

Chomczynski P, Mackey K. (1995) Short technical report. Modification of the TRIZOL reagent procedure for isolation of RNA from Polysaccharide-and proteoglycan-rich sources.Biotechniques 19(6): 942-5.