RNA purification --- guanidinium thiocyanate

1. Prepare the guanidinium thiocyanate stock solution ( 4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7.0, 0.5 % Sarkosyl )

Add beta-mercaptoethanol to the final concentration of 0.1 M just before use.

2. Lysis cells or tissue samples in the guanidinium thiocyanate stock. For cells, add directly to cell pellet and immediate freeze in dry ice/ethanol or liquid nitrogen. Lysis cells by freezing and thawing three times. For tissues, homogenize with a homogenizer (e.g., mortar and pestle, grind to a fine powder). Put sample on ice.

3. Add 1/10 volume of 2 M sodium acetate (pH 4.0). Vortex.

4. Add equal volume of water saturated phenol. Vortex.

5. Add 1/10 volume of chloroform:isoamyl alcohol (49:1) (phenol:chloroform:isoamyl alcohol 250: 49: 1). Vortex for 10 seconds.

6. Let sit on ice for 15 minutes.

7. Centrifuge at 4 C for 20 minutes to separate the aqueous phase.

8. Transfer the top phase to a fresh tube.

9. Repeat phenol:chloroform:isoamyl alcohol extraction once.

10. Add 1 volume of cold isopropanol and precipitate at -20 C for at least 1 hour.

11. Pellet RNA by centrifugation for 20 minutes at 4 C.

12. Wash the pellet with 70% ethanol.

13. Air dry. Resuspend RNA in RNAse-free TE or ddH2O.

14. For long term storage, add 1/10 volume of 3 M sodium acetate (pH5.2) (DEPC-treated) and 2.5 volume of 100% ethanol, store at -20 C.