Total RNA Isolation

1.0 g plant tissue:

1. grind tissue with pestle and mortar in liquid nitrogen
2. transfer to a 50 ml falcon tube containing 10 ml of sol D
3. add 1 ml 2 M NaOAc pH 4.1
4. add 10 ml phenol
5. add 2 ml chloroform/isoamylalcohole (49:1)
6. mix thoroughly
7. shake finally for 10 sec
8. cool on ice for 15 min
9. spin at 3500 rpm/4°C for 20 min in an Omnifuge 2.0 RS centrifuge (Heraeus)
10. transfer aqueous phase to a fresh 30 ml corex glass tube
11. add 10 ml isopropanole
12. precipitate for 1 hour at -20°C
13. spin at 8000 rpm/4°C for 20 min in a HB-4 rotor
14. dissolve RNA pellet in 3 ml sol D (ev. heat to 60°C to dissolve pellet)
15. add 3 ml isopropanole and precipitate for 1 hour at -20°C
16. spin at 8000 rpm/4°C in a HB-4 rotor for 15 min
17. resuspend pellet in freshly prepared 75% EtOH
18. repeat centrifugation, remove supernatant and air dry for 10-15 min
19. dissolve pellet in 3-5 ml of sterile, freshly autoclaved H₂O (ev. heat to 60°C to dissolve pellet)

Solutions:

Guanidium thiocyanate sol:

100.0 g GTC

117.2 ml H₂O

7.04 ml 0.75 M NaZit pH 7

10.56 ml 10% sarcosyl

220 ml total

Sol D:
add 0.36 ml b-mercaptoethanole/50 ml GTC sol (1.6ml/220 ml), stable for 1 month at RT

2 M NaOAc pH 4.1 (100 ml):

dissolve 27.2 g NaOAc in about 54 ml H₂O and add about 46 ml conc HAc

Remarks:

As usual when working with RNA: pay attention, always wear gloves, use only freshly prepared and autclaved material and use sterile plastic pipettes. In our experience there is no need for DEPC-treated material.

Corex tubes and pestle and mortar have to be pretreated with H₂O₂ for 30 sec, rinsed several times with H₂O and baken for 6 hours at 180°C in oven.

Materials:

Reagent	Supplier	Cat.-#
guanidium thiocyanate	Sigma	G-9277
isopropanole	Fluka	59300
NaOAc	Fluka	71180
NaZit	Sigma	S-4641
sarcosyl	Sigma	L-9150
b-mercaptoethanole	Sigma	M-6250