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Protocol for RNA Isolation:

 

1. Homogenization

 

a. Tissues

Homogenize tissue samples in 1 ml of TRIZOL reagent for a piece of tissue no more than 100mg by mortar and pestle. Cover the tissue sample with liquid nitrogen. Note: when handling liquid nitrogen, wear safety mask and other appropriate gear. And proceed to smash the tissue until it has reached a fine, powder-like texture. Alternatively, one can use a glass-Teflon or manual homogenizer. The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization.

 

b. Cells Grown in monolayer

Lyse cells directly in a culture in a culture dish by adding 1 ml of TRIZOL Reagent to 3.5-cm diameter dish, and passing the cell lysate several times through a pipette. 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 contamination of the isolated RNA with DNA.

 

c. Cells Grown in Suspension

Pellet cells by centrifugation. Lyse cells in TRIZOL reagent by repetitive pipeting. Use 1 ml of the reagent per 5-10x10­­ cells. Washing cells before addition of TRIZOL Reagent should be avoided, as this increases the possibility of mRNA degradation.

 

2. Phase separation

 

Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIZOL reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 15 minutes. Following centrifugation at 2 to 8°C, the mixture separates into a lower red, phenol-chloroform phase (an interphase), and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIZOL reagent used for homogenization.

 

3. RNA precipitation

 

Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. 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 30°C for 10 minutes and centrifuge at no more than 12,000xg for 10 minutes at 2 to 8°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.

 

4. RNA wash

 

Remove the supernatant. 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 sample by vortexing, then centrifuge at no more than 7,500 g for 5 minutes at 2 to 8°C.

 

5. Re-dissolving the RNA

 

At the end of the procedure, briefly dry the RNA pellet (air-dry or vacuum-dry) for 5-10 minutes at 55 to 60 °C.