Isolation of RNA from Animal Tissue

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1. Rinse tissue in ice-cold PBS Heparin.

2. Put tissue into Guanidinium Thiocyanate Solution (see Hint #2). Homogenize with a polytron homogenizer until the tissue has completely dissipated (i.e., until you cannot see any pieces of tissue remaining).

3. If using 8 ml of Guanidinium Thiocyanate Solution, layer the homogenate over a 3 ml CsCl Solution in a SW41 polyallomer tube. If using 3 ml of Guanidinium Thiocyanate Solution, layer the homogenate over a 1.5 ml CsCl Solution in SW50.1 polyallomer tubes.

4. Centrifuge in the appropriate rotor at 20°C for 20 hr. Use the SW41 rotor at 32,000 rpm (130,000 X g) and the SW50.1 rotor at 36,000 rpm (96,000 X g).

5. Remove all of the supernatant (the RNA will be a clear pellet). Wipe the inside of the tube with a lab tissue, avoiding the RNA pellet.

6. Suspend the pellet in 250 μl DEPC-treated ddH2O (see Hint #3) and transfer to a microcentrifuge tube. Rinse the centrifuge tube with an additional 250 μl DEPC-treated ddH2O and add it to the microcentrifuge tube containing the RNA pellet. Vortex, then heat-shock the tube contents by incubating the tube at 65°C for exactly 3 minutes. Mix by vortexing and examine the tube to make sure the RNA is dissolved.

7. Add an equal volume of Buffer-Equilibrated Phenol (CAUTION! See Hint #1; also see Protocol ID#578).

8. Mix by vortexing.

9. Centrifuge in a microcentrifuge at full speed for 10 min.

10. Transfer the upper aqueous phase to a fresh tube. Avoid any of the interface.

11. Add 0.5 volume of Buffer-Equilibrated Phenol and mix by vortexing.

12. Add 0.5 volume Chloroform (CAUTION! See Hint #2) and mix by vortexing.

13. Centrifuge 2 min at full speed in a microcentrifuge. Recover the aqueous phase, repeat the Chloroform extraction, and recover the aqueous phase.

14. Precipitate the RNA by adding 0.1 volume of 3.0 M Sodium Acetate and 2 volumes of Ethanol. Freeze the sample in Dry Ice, or at -70°C, or overnight at -20°C.

15. Centrifuge in a microcentrifuge at full speed for 10 to 15 min. Resuspend in DEPC-treated water.

16. Read the absorbance at 260 nm and 280 nm.

17. Check for RNA integrity by electrophoresis .


Solutions

PBS Heparin  pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
Add 10 Units/ml Heparin
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl


CsCl Solution  10 mM EDTA, pH 8.0
5.7 M CsCl
Density should be between 1.7 and 1.72 g/ml
25 mM Sodium Acetate, pH 5.0
Filter


Guanidinium Thiocyanate Solution  4.0 M Guanidinium Thiocyanate (CAUTION! See Hint #1)
Add 0.1% Antifoam A
adjust to pH 7.0 with 1.0 M NaOH
0.5% Sarkosyl
25 mM Sodium Citrate, pH 7.0
filter through Whatman paper
0.1 M 2-Mercaptoethanol


BioReagents and Chemicals

Sodium Acetate
Ethanol
2-Mercaptoethanol
Sarkosyl
Potassium Phosphate, Monobasic
Diethyl Pyrocarbonate (DEPC)
Sodium Hydroxide
Cesium Chloride
Heparin
Antifoam A
Chloroform
Phenol
Dry Ice
Guanidinium Thiocyanate
EDTA
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Citrate
Sodium Chloride

Protocol Hints

1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Use 8 ml for large mouse tissues such as liver, kidneys, brain, etc. Use 3 ml for smaller tissues such as testis, bone marrow, etc.

3. See BioTools section.