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 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 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.