Materials

• GIT (4M Guanidine Isothiocyanate in 0.025 M Sodium Acetate (pH 6). Add 8.3 µl ß-MercaptoEthanol (2-ME) for each ml GIT immediately before use).
  
• CsCl (5.7 M CsCl in 0.025 M Na Acetate pH 6).
  
• SEVAG (Chloroform:Isoamyl Alcohol - 24:1 Volume:Volume)

1. Homogenize 0.1-0.2 gm tissue (frozen or fresh) or up to 10⁸ cells in 2.5 ml GIT with 2-ME.
   
2. Layer GIT homogenate onto 2 ml CsCl in 6 ml tube for SW50.1 rotor. Centrifuge 35,000 RPM/20 hours/20°C.
   
3. Aspirate and discard upper GIT layer containing denatured proteins. Upper 1 ml portion of CsCl can be saved for DNA preparation.
   
4. Aspirate and discard all remaining liquid with care - do not contaminate RNA pellet.
   
5. Break up the RNA pellet with a barrier pipette tip and THEN dissolve in 0.2 ml NaOAc (0.3 M) and transfer to microfuge tube.
   
6. Extract 2-4X with PHENOL/SEVAG pH 4-6 (1:1 Ph:S)
   [note - if phenol alone is preferred the pH must be above 7 and yields may be lower].
   
7. Extract 2X with SEVAG Ethanol Precipitate and store in ethanol at -20°C.

Method

1. Vigorously pipette 5-10 x10⁶ cells or homogenize 50-100mg tissue in 1 ml TRIzol®. Let stand 5 minutes to permit the complete dissociation of nucleoprotein complexes.
2. Add 0.2 ml Chloroform and shake by hand for 15 seconds - stand 3 minutes.
3. Centrifuge 12,000 x g, 15 minutes, 4°C.
4. Transfer supernate to new tube and add 0.5 ml isopropanol.
5. Mix and incubate 10 minutes at room temperature. Centrifuge 10 minutes, 12,000 x g, 4°C.
6. Discard supernate and rinse pellet with 1 ml 75% ethanol.
7. Centrifuge 7500 x g, 5 minutes, 4°C. Remove all supernate and air dry briefly.
8. Resuspend pellet with TE. If necessary, heat briefly ~60°C to dissolve pellet completely.
9. Add 1/10 volume 3 M NaOAc and 3 volumes ethanol and store at -70°C.
10. Note: Genomic DNA may also be prepared during either of the RNA procedures described here.