Purification of RNA with TRIzol reagent

TRIzol reagent is a mixuture of phenol and guanidine isothiocyanate suitable for small-scale extraction of RNA. (TRIzol reagent is a registered trademark of Molecular Research Center, Inc.)


1. Grind 30-100 mg of frozen tissue in a prechilled microfuge tube with prechilled micropestle. Keep the tissue frozen with liquid nitrogen.

2. Add 600 ul of TRIzol reagent (Invitrogen) and grind at room temperature until no visible debris remains.

3. Store the tube on ice until all of the samples are ground.

4. Add 400 ul of TRIzol reagent to each tube. Store the samples for 5 minutes at room temperature.

5. Add 0.2 ml of chloroform per 1 ml of TRIzol reagent. Shake the tube vigorously for 15 seconds.

6. Spin for 15 minutes at 4 C. Transfer the aquaous phase to a new tube.

7. Add 250 ul of isopropanol and 250 ul of precipitation solution (0.8 M sodium citrate, 1.2 M NaCl). Mix well. Stand the tubes for 10 minutes at room temperature.

8. Spin for 10 minutes at 4 C. Discard the supernatant.

9. Rince the RNA pellet with 1 ml of 75% ethanol. Spin for 5 minutes at 4 C. Discard the supernatant.

10. Dry the pellet for 10 minutes. Dissolve the RNA in RNase-free water.

11. Store at -70 C.

Typical yield is 20-50 ug of RNA from 100 mg of Arabidopsis tissue. However, yield of silique RNA is very low with this procedure in our hands. For silique, follow the protocol described bellow.

Extraction of RNA from silique

1. Make 1:1 mixture of extraction buffer (1M Tris-Cl (pH 9.0), 1% SDS) and phenol-chloroform. Phenol-chloroform is made by mixing equal volumes of TE-saturated phenol and chloroform just before use.

2. Add 30-100 mg of frozen silique into 600 ul of the above mixture in microfuge tube.

3. Grind the silique with micropestle.

4. Vortex 30 seconds, and then spin at 14,000 rpm for 5 minutes at 4 degrees. Transfer the aquaous phase into new tube.

5. Extract once more with phenol-chloroform, and then extract 3 times with TE-saturated phenol.

6. Add 1/10 volume of 3M NaOAc and 3 volumes of ethanol. Spin for 10 minutes at 14,000 rpm at 4 degrees. Discard the supernatant.

7. Rinse the pellet with 1 ml of 70% ethanol. Spin for 5 minutes. Discard the supernatant.

8. Dissolve the pellet in 150 ul of RNase-free water. Add 50 ul of 8M LiCl. Mix well.

9. Store on ice overnight.

10. Spin at 14,000 rpm for 10 minutes at 4 degrees. Discard the supernatant.

11. Rinse the pellet with 200 ul of 2M LiCl. Spin for 5 min at 14,000 rpm. Discard the supernatant.

12. Rinse the pellet with 500 ul of 70% ethanol. Spin for 5 min at 14,000 rpm. Discard the supernatant.

13. Air-dry the pellet for 10 minutes. Dissolve the RNA pellet in RNase-free water.

Typical yield is 20-50 ug of RNA from 100 mg of Arabidopsis silique.



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