RNA EXTRACTION |
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Last Update: December 2006 |
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This protocol is based on the protocol developed by Pamela Green's laboratory and the protocols described in Biotechniques 8:148-149 (1990) and Biotechniques 19: 734-737 (1995) |
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| PREPARE SOLUTIONS | |
| 1. GTC buffer (400 mL): | Mix 189.1 g of guanidium thiocyanate (FW: 118.16, 4M), 10 mL of 1M sodium citrate, pH 7 (25 mM), 2 g of sarkosyl (0.5%), and 2.8 mL of 14.3M betamercaptoethanol (0.1 M) |
| 2. Pellet resuspension solution (200 mL): | Mix 5 mL of 20% SDS (0.5%), 2 mL of 1M Tris-HCl
pH 7.5 (10 mM), and 0.4 mL of 0.5 M EDTA (1 mM) |
| PROCEDURE - STANDARD EXTRACTION | |
| 1. Grind material (1 gm) under liquid nitrogen | |
| 2. Resuspend the powder in 5 mL of GTC buffer in a 13 mL round bottom screw-cap Sarstadt tube | |
| 3. Add 0.5 mL 2M sodium acetate pH 4.0 and vortex | |
4. Add 5 mL of phenol-TE and vortex |
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| 5. Add 1 mL of chloroform and vortex | |
| 6. Spin for 10 mins at 10,000 xg | |
| 7. Save upper phase and transfer to new 13 mL tube containing 5 mL of isopropanol | |
| 8. Place on ice for 10 mins or ON at -20oC to precipitate RNA | |
9. Spin for 10 mins at 10,000 xg |
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| 10. Transfer pellet to a 2 mL microcentrifuge tube by adding 1 mL 4 M LiCl2. DO NOT VORTTEX. The pellet should dislodge easily with the addition of the LiCl2. Rinse tube with an additional 0.5 mL LiCl2 to remove any left over pellet and combine all this in the 2 mL tube | |
| 11. Place all 2 mL tubes in a multiple vortex pad and vortex for 10-15 mins until pellet is completely resuspended | |
| 12. Spin in microfuge for 5 mins to pellet RNA | |
| 13. Add 1 mL LiCl2 to pellet and repeat vortex and 5 mins spin | |
| 14. Resuspend pellet in ~0.7 mL pellet resuspension solution. Make sure the entire pellet is resuspended (vortex at least 5 mins) | |
15. Sequentially extract the aqueous phase with equal volumes of phenol, phenol/chloroform, and 24:1 chloroform/isoamyl alcohol |
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| 16. Quantify RNA concentration | |
17. Store at -70oC |
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| PROCEDURE - RNA extraction from samples with high contents of polysaccharides | |
1. Prepare Extraction buffer (100 mL ): Mix 10 mL of 1M Tris-HCl pH 9.0 (100 mM), 5 mL of 4N NaCl (200 mM), 3 mL of 0.5 M EDTA (15 mM), 0.5 g of sarkosyl (0.5%), and 83 mL of dH2O. Add 8 mL/mL of b-MetSH prior to use |
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| 2. Grind tissue in a mortar in liquid nitrogen (0.5-1 g of fresh tissue) | |
3. Transfer to a tube containing 4 mL extraction buffer with b-MetSH |
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| 4. Add 4 mL of phenol and vortex | |
5. Add 0.8 mL 24:1 Chloroform:isoamul alcohol and vortex |
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| 6. Add 280 mL 3 M sodium acetate pH 5.3 and vortex | |
| 7. Chill for 15 mins on ice | |
8. Spin for 10 mins at 10,000 xg |
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| 9. Add 1 volume of phenol:chloroform: isoamyl alcohol and vortex | |
10. Spin for 10 mins at 10,000 xg |
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11. Optional: repeat step 9 and 10 |
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12. Transfer aqueous phase to a new tube and add 1 volume of isopropanol |
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13. Chill at -70oC for 20 mins |
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14. Spin for 10 mins at 10,000 xg |
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15. Wash with 80% EtOH |
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16. Spin for 10 mins at 10,000 xg |
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17. Dry pellet |
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18. Resuspend in 1 mL DEPC-dH2O |
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19. Transfer to a 2 mL tube. Spin down for 3 mins to remove insoluble material |
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20. Adjust to 1 mL with dH2O and add 0.5 mL of 8 M LiCl2 (2.6M final concentration) |
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21. Precipitate RNA for 2-3 hours on ice or ON |
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22. Spin for 10 mins at 10,000 xg |
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23. Optinal: wash pellet with 2 N LiCl2 |
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24. Wash with 80% EtOH and Spin for 10 mins at 10,000 xg |
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25. Dry sample and resuspend RNA in DEPC-dH2O (50-200 mL depending on pellet size) |
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26. Spin if necessary (if there are insoluble materials) |
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27. Quantify RNA concentration |
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28. Store at -70oC |
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