EXTRACTION AND PURIFICATION OF TOTAL RNA USING TRIzol OR TRI REAGENT
Susan L. Gottshall, Saban Tekin, and Peter J. HansenDept. of Animal Sciences, University of Florida (SLG is currently at Purdue Pharma Inc.)
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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.
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 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.
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 μl 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 μl 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 μl RNA sample from the original stock, diluted with 998 or 995 μl 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 μg/μl]= 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)
Preparation of RNase-free water
a. Measure water into RNase-free glass bottles.
b. Add 1 ml of 0.1% (v/v) diethylpyrocarbonate (DEPC) to 1000 ml water.
c. Let stand overnight.
Note: RNase free DEPC treated water is Biotecx brand (cat # BL-5611).
Note: If using 0.5% SDS solution to resuspend the RNA. It must be prepared in RNase free water.
Website maintained by Peter J. Hansen Modified 05-15-2008