RNA Extraction from RNase-Rich Senescing Leaf Samples

Materials and Reagents

1. Guanidinium thiocyanate (Thermo Fisher Scientific, catalog number:  BP221 )
2. NaCitrate
3. N-Lauroylsarcosine (Sigma-Aldrich, catalog number: L9150 )
4. β-mercaptoethanol (β-ME)
5. Glacial HAc
6. NaOH
7. Phenol (J.T.Baker^®, catalog number:  2859 )
8. Ethylenediaminetetraacetic acid (EDTA)
9. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
10. Tris (hydroxymethyl) aminomethane
11. Chloroform
12. Isopropanol
13. Diethylpyrocarbonate (DEPC)
14. Ethanol
    
15. Extraction buffer (EB) (see Recipes)
    
16. 2 M NaAcetate (pH 4.0) (see Recipes)
17. 2 M NaAcetate (pH 5.0) (DEPC treated) (see Recipes)
    
18. H₂O-saturated phenol (no buffer required) (see Recipes)
    
19. Citrate-EDTA-SDS solution (CES) (see Recipes)
    
20. Tris-EDTA-SDS solution (TES) (see Recipes)
    
21. 100 ml 0.75 M NaCitrate (see Recipes)
    
22. 60 ml 10% Sarkosyl (see Recipes)
    

Equipment

1. Vortex Mixer 
2. Beckman high speed centrifuge with JS13.1 rotor or the like
3. 15-ml Corning centrifuge tube
   

Procedure

1. 0.2-0.5 g sample tissue ground in liquid N₂ (better grinding ensures a higher RNA yield).
2. 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.
3. Add 0.5 ml 2 M NaAcetate (pH 4.0) to the tube, vortex 1 min at room temperature.
4. Add 5 ml H₂O-saturated phenol, vortex 1 min at room temperature.
5. Add 1 ml chloroform, vortex 1 min at room temperature.
6. 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.
7. 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.
8. Spin 10 min at 3,000 x g, 4 °C (4,500 rpm JS13.1), discard supernatant.
9. Add 2 ml CES or TES to redissolve pellet.
10. Add 2 ml chloroform, vortex 1 min at room temperature.
11. Spin 10 min at 3,000 x g, 4 °C, transfer aqueous to new tube.
12. 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.
13. 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

1. 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. 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
3. 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
4. H₂O-saturated phenol (no buffer required)
5. 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

6. Tris-EDTA-SDS solution (TES)
   10 mM Tris (pH 7.5)
   1 mM EDTA (pH 8.0)
   0.5% SDS   [DEPC]
7. 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
8. 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

1. 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.
2. 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.