Protocols

1.

Remove all the culture supernatant from cell pellet or cell monolayer and add Solution D/ß-ME (600 µl for < 2 x 10⁷ cells). If cell numbers are low (<10⁶), 5 µg yeast tRNA ca be added as a carrier. Mix thoroughly by pipetting and transfer to a 1.5 ml eppendorf tube. For higher cell numbers, use a larger size polypropylene tube or Corex tube.

2.

Add 0.1 volume of 2 M sodium acetate, pH 4.0, to tube and mix.

3.

Add 1 volume of water-saturated phenol to tube, mix.

4.

Add 0.2 volume of chloroform:isoamyl alcohol (49:1) to tube.

5.

Vortex vigorously until an emulsion forms and incubate tube for 15 minutes on ice.

6.

Centrifuge at 12,000 x g for 20 minutes at 4°C

7.

Carefully transfer maximum volume of the upper aqueous phase to a new tube but avoid any contact with the interphase (save the interphase and organic phase for protein extraction). Add an equal volume of isopropanol, mix by inversion, and incubate at -20°C for at least 1 hour.

8.

Centrifuge at 12,000 x g for 20 minutes at 4°C and remove supernatant.

9.

Resuspend the pellet in Solution D/ß-ME (600 µl) by gentle vortexing. Add equal volume of isopropanol. Mix by inversion and incubate at 20°C for at least 1 hour.

10.

Centrifuge at 12,000 x g for 20 minutes at 4°C. Carefully remove the supernatant and wash the pellet twice with ~ 1 ml 66% ethanol. Air dry pellet and dissolve RNA in RNase-free H₂O at 65°C for 10 minutes. To determine the concentration and purity of RNA, dilute 2-10 µl of RNA in 0.6 ml H₂O, check absorption at 260 and 280 nm. The A260:A280 ratio should be around 2.0. For RNA the OD₂₆₀ unit is equal to 30 µg/ml. Store the RNA at -70°C.