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.

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 0.01% (v/v) diethylpyrocarbonate (DEPC).

c.Let stand overnight.

d.Autoclave.

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.