Materials and Reagents

Guanidinium thiocyanate (Thermo Fisher Scientific, catalog number: BP221 )
NaCitrate
N-Lauroylsarcosine (Sigma-Aldrich, catalog number: L9150 )
β-mercaptoethanol (β-ME)
Glacial HAc
NaOH
Phenol (J.T.Baker^®, catalog number: 2859 )
Ethylenediaminetetraacetic acid (EDTA)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
Tris (hydroxymethyl) aminomethane
Chloroform
Isopropanol
Diethylpyrocarbonate (DEPC)
Ethanol
Extraction buffer (EB) (see Recipes)
2 M NaAcetate (pH 4.0) (see Recipes)
2 M NaAcetate (pH 5.0) (DEPC treated) (see Recipes)
H₂O-saturated phenol (no buffer required) (see Recipes)
Citrate-EDTA-SDS solution (CES) (see Recipes)
Tris-EDTA-SDS solution (TES) (see Recipes)
100 ml 0.75 M NaCitrate (see Recipes)
60 ml 10% Sarkosyl (see Recipes)

Equipment

Vortex Mixer
Beckman high speed centrifuge with JS13.1 rotor or the like
15-ml Corning centrifuge tube

Procedure

0.2-0.5 g sample tissue ground in liquid N₂ (better grinding ensures a higher RNA yield).
Add to 15-ml Corning centrifuge tube containing 5 ml EB (2.5 ml EB if sample is less than 0.2 g), vortex 2 min at room temperature.
Add 0.5 ml 2 M NaAcetate (pH 4.0) to the tube, vortex 1 min at room temperature.
Add 5 ml H₂O-saturated phenol, vortex 1 min at room temperature.
Add 1 ml chloroform, vortex 1 min at room temperature.
Spin 10 min at 10,000 x g, 4 °C (8,000 rpm, JS13.1 rotor), transfer aqueous phase to a new 15-ml Corning centrifuge tube.
Add equal vol. isopropanol to the new tube, screw cap and mix by inverting several times, keep the tube at -20 °C for >1 h.
Spin 10 min at 3,000 x g, 4 °C (4,500 rpm JS13.1), discard supernatant.
Add 2 ml CES or TES to redissolve pellet.
Add 2 ml chloroform, vortex 1 min at room temperature.
Spin 10 min at 3,000 x g, 4 °C, transfer aqueous to new tube.
Add 1/10 vol. 2 M NaAcetate (pH 5.0), equal vol. isopropanol, pellet by spinning 10 min at 3,000 x g, 4 °C.
Wash the pellet with 75% and 100% ethanol, respectively, air dry the pellet, and dissolve the pellet with 50-200 μl DEPC-treated CES or TES (for RT PCR or other analyses involving enzymes, DEPC-treated CE or TE without SDS should be used). The total RNA yield from senescent leaves is low, so a reduced final volume is suggested.

Recipes

EB in 100 ml in 300 ml

4 M Guanidinium thiocyanate (MW 118.2)	47.28 g	141.84 g
25 mM NaCitrate (pH 7.0) (MW 294.10)	3.33 ml of 0.75 M	10 ml of 0.75 M
0.5% Sarkosyl (N-Lauroylsarcosine, 293.4)	5 ml of 10% Soln	15 ml of 10%
0.1 M β-ME (in 5 ml EB, add 35 μl conc)	53.8 ml H₂O

2 M NaAcetate (pH 4.0) (MW 82.03)
11.49ml glacial HAc (17.4 M)
70 ml H₂O
Titrate w/ NaOH to pH 4.0 (this pH value is essential)
ddH₂O to 100 ml
2 M NaAcetate (pH 5.0) (MW 82.03) (DEPC treated)
11.49ml glacial HAc (17.4 M)
70 ml H₂O
Titrate w/ NaOH to pH 5.0
ddH₂O to 100 ml
H₂O-saturated phenol (no buffer required)
Citrate-EDTA-SDS solution(CES) for 100 ml
10 mM NaCitrate(pH 7.0)
1.333 ml 0.75 M NaCitrate (pH 7.0)
(DEPC) 1 mM EDTA (pH 8.0)
200 μl 0.5 M EDTA
0.5% SDS
2 ml 25% SDS
Tris-EDTA-SDS solution (TES)
10 mM Tris (pH 7.5)
1 mM EDTA (pH 8.0)
0.5% SDS [DEPC]
100 ml 0.75 M NaCitrate
15.761 g Citric acid (MW 210.14)
80 ml ddH₂O
Titrate w/ NaOH to pH 7.0
ddH₂O to 100 ml
60 ml 10% Sarkosyl
6 g N-lauroylsarcosine (MW 293.4)
60 ml ddH₂O
65 °C stir

Acknowledgments

This protocol was initially developed from Chomczynski and Sacchi (1987) and Puissant and Houdebine (1990) at the Amasino Lab, University of Wisconsin-Madison and finalized at Cornell University. Su-Sheng Gan was a recipient of The Rockefeller Foundation Biotechnology Predoctoral Fellowship. The research was also partially supported by a Federal Formula Fund at Cornell University. The past and current members of the Gan Lab also greatly helped in improving the protocol.

References

Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1): 156-159.
Puissant, C. and Houdebine, L. M. (1990). An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Biotechniques 8(2): 148-149.