EXTRACTION AND PURIFICATION OF TOTAL RNA USING TRIzol

Friday, October 17, 2003

Description
RNA Extraction using Trizol reagent

Procedure
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 oC.

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 l. If you using 1.5 -2 ml microcentrifuge tube and pestle for homogenization, start with 500 l of TRIzol or TRI reagent, then add remaining 500 l.

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.

3. RNA Precipitation

a. Transfer the upper aqueous phase to a fresh tube.

b. Add 0.5 ml of isopropyl alcohol to precipitate RNA. If this RNA will be used for RT-PCR, first add 50 l isopropyl alcohol, mix, incubate the samples at room temperature for 5 minutes and centrifuge at 12,000 x g for 10 minutes at 4oC. Transfer the sample in a new tube.

c. Incubate for 5-10 minutes at room temperature.

d. Centrifuge for 10 minutes at 12,000 x g at 4oC. The RNA will form a pellet on the side or bottom of the tube.

4. RNA Wash

a. Discard the supernatant.

b. Wash pellet with 1 ml 75% ethanol.

c. Mix sample by vortexing. The RNA pellet may float.

d. Centrifuge at 12000 x g for 5 minutes at 4oC. If this RNA will be used for RT-PCR repeat steps a,b and c twice.

e. RNA pellet may be stored in ethanol at -70oC for months.

5. Redissolving the RNA

a. Remove supernatant.

b. Air dry the pellet for 5-10 minutes. Do not completely dry out the pellet.

c. Dissolve pellet in 30 to 60 l RNase free water or 0.5% SDS by passing the solution through a pipette tip and incubating for 10 minutes at 55-60oC.

6. Determination of RNA Concentration and Purity

a. Take 2 to 5 l RNA sample from the original stock, diluted with 998 or 995 l RNase free water in a 1.5 ml microcentrifuge tube. This will give you 500 or 200 time dilution of the RNA sample.

b. Pipet 1 ml RNAse free water in a clean cuvette and read absorbance as blank.

c. Pipet the diluted RNA sample in to a clean cuvette and read absorbance at 260 nm and 280 nm.

d. Use the formula below to determine RNA Concentration of the original sample:

[RNA g/l]= A260 x 33 x dilution factor / 1000

e. To determine the purity of the RNA sample, calculate ratio of A260/A280. Ratios between 1.7 to 2

represent good RNA)

Recipes
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

Supplies

Tips

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EXTRACTION AND PURIFICATION OF TOTAL RNA USING TRIzol