RNA purification using TRizol--TRIZOL法提取RNA

Reagents and Equipments

TRIzol Reagent (Life Technologies cat# 15596-026) or TRI reagent (Sigma cat # T-9424)

DEPC (RNase free) water or 0.5% SDS solution in DEPC treated water

Chloroform (Fisher )

Isopropyl alcohol (2-Propanol) (Fisher)

75% Ethanol (in DEPC treated water)

Sterile or RNase treated pipette tips, microcentrifuge tubes, and pestles or motorized homogenizer.

Microcentrifuge

1.Homogenization for Cell Suspensions

a.Place 1 ml aliquots of the cell suspension in sterile RNase free 1.5 ml microcentrifuge tubes.

b.Centrifuge for 1 minute to pellet the cells.

c.Pour off the supernatant.

d.Add 1 ml of TRIzol or TRI reagent to the tubes.

e.Lyse cells by repetitive pipetting.

f.Centrifuge homogenate at 12000 x g for 10 minutes at 4℃.

g.Transfer the homogenate in a sterile microcentrifuge tube.

h.If this RNA will be used for RT-PCR, repeat steps f and g twice.

Modification for tissues

a.Add 1 ml of TRIzol or TRI reagent to every 50-100 mg of tissue. Sample volume should not exceed 100 μl. If you using 1.5 -2 ml microcentrifuge tube and pestle for homogenization, start with 500 μl of TRIzol or TRI reagent, then add remaining 500 μl.

b.Homogenize the samples using a sterile or RNAse free plastic, glass pestles or power homogenizer and tubes.

Modification for monolayers

a.Lyse cells directly in a culture dish by adding 1 ml of TRIzol or TRI reagent to a 3.5 cm diameter dish.

b.Pass the cells through a pipette several times.

2. Phase Separation

a.Incubate samples (from 1g) for 5 minutes at room temperature.

b.Add 0.2 ml of chloroform to each tube.

b.Cap each tube. Shake samples vigorously by hand for 15 seconds.

c.Incubate samples for 5 minutes at room temperature.

d.Centrifuge samples for 15 minutes at 12,000 x g at 4oC.