Molecular Techniques and Methods

Isolation of Mitochondrial RNA from Plant Tissues

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The mtRNA constitutes about 1% of the total RNA of plants. Of this, at least 75% is of ribosomal origin.

In this procedure, mitochondria are separated from other subcellular components by differential centrifugation of a tissue homogenate. Sedimentation through sucrose gradients yields a mitochondrial fraction substantially free of other organelles. The purified mitochondria are lysed in the presence of a nuclease inhibitor ATA (aurintricarboxylic acid), and the lysate is extracted with organic solvents (phenol: chloroform) to remove protein and other contaminants. Finally, the nucleic acids are concentrated by precipitation (in ethanol or LiCl).

  • A typical yield would be 0.5-1 ug mtRNA per gram fresh weight of etiolated shoots.





  • MATERIALS AND SOLUTIONS

    Plant Material
    For etiolated shoots and green leaves, 20 g tissue should normally be considered a minimum requirement. If the tissue (e.g., maize kernel scutella and pea buds) has a high cell density, a few grams may suffice. The plant material should be free of visible fungal and bacterial contamination, but low levels will not interfere with the procedure. Bacteria may copurify with the mitochondria.


    Isolation Buffer (1 liter)
    0.35 M Sorbitol ------------------------------------------ 63.8 g
    50 mM Tris-HCl (pH 8.0) -------------------------------- 50 ml of 1 M Tris-HCl
    5 mM EDTA --------------------------------------------- 10 ml of 0.5 M EDTA
    0.1% Bovine Serum Albumin ----------------------------- 1 g
    Spermine ------------------------------------------------- 0.25 g
    Spermidine ----------------------------------------------- 0.25 g
  • Add DEPC-treated H2O to make a final volume of --- 1 liter
  • Store at 4oC.
  • Add 1.25 ml of 2-mercaptoethanol just before to use.


    30%, 52%, and 60% Sucrose Gradient Buffer
    Dissolve sucrose in Wash Buffer


    Wash Buffer (1 liter)
    0.35 M Sorbitol --------------------------------------- 63.8 g
    50 mM Tris-HCl (pH 8.0) ----------------------------- 50 ml of 1 M Tris-HCl
    20 mM EDTA ----------------------------------------- 40 ml of 0.5 M EDTA
    Add DEPC-treated H2O to make a final volume of --- 1 liter
  • Store at 4oC.


    Lysis Buffer (10 ml)
    10% (w/v) Sodium sarkosyl ---------------------------- 1 g
    25 mM Tris-HCl (pH 7.5) ------------------------------ 0.25 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------------ 0.4 ml of 0.5 M EDTA
  • Add DEPC-treated H2O to make a final voluem of --- 10 ml
  • Store at 4oC.


    100 mM ATA (AurinTricarboxylic Acid) Stock Solution (1 ml)
    100 mM AurinTricarboxylic Acid (Ammonium salt, Sigma) --- 47.3 mg
    50 mM Tris-HCl (pH 8.0) ----------------------------------- 50 ul of 1 M Tris-HCl
    DEPC-treated H2O to make a final volume of ------------- 1 ml
  • Keep in foil-covered bottles at 4oC.
  • Use at a concentration of 1 mM during lysis of mitochondria and at 50 uM for long-term storage of RNA.


    Storage Buffer (10 ml)
    25 mM Tris-HCl (pH 8.0) ------------------------------ 250 ul of 1 M Tris-HCl
    50 uM ATA -------------------------------------------- 5 ul of 100 mM ATA
    DEPC-treated H2O ------------------------------------ 9.745 ml
  • Store at 4oC.


    Acid Phenol (pH 4.5) containing 0.1% 8-hydroxyquinoline

    Chloroform: IAA (24:1)




    PROCEDURES

    Isolation of Mitochondria

  • All manipulations should be performed on ice! Always wear gloves!

    1. Wash roots, tubers, or immature fruits thoroughly with sterile water.

    2. Cut etiolated shoots, green leaves, or roots into small segments with scissors.

    3. Homogenize 20 g tissue in 40 ml of ice-cold Isolation Buffer by Waring blender. Two times for 10 seconds at low speed and one time for 5 seconds at high speed.

    4. Homogenize again with a polytron (Brinkman Instruments). Two times for 10 seconds at high speed.

    5. Filter the homogenate through four layers of cheesecloth and one layer of Miracloth into centrifuge bottles in ice.

    6. Centrifuge the filtrate for 10 minutes at 1,000g. (The pellet contains cellular debris, plastids, starch, and nuclei.)

    7. Decant the supernatant into centrifuge bottles in ice.

    8. Centrifuge for 20 minutes at 8,500g to collect mitochondria.

    9. Resuspend crude mitochondrial pellet in 10 ml of ice-cold Isolation Buffer with a paintbrush. It is essential that the mitochondria are completely dispersed.

    10. Add additional 90 ml ice-cold Isolation Buffer.

    11. Centrifuge the suspension again for 10 minutes at 1,000g.

    12. Decant the supernatant carefully into centrifuge bottles in ice.

    13. Centrifuge for 20 minutes at 8,500g to collect mitochondria.

    14. Discard the supernatant.

    15. Resuspend the final mitochondrial pellet in 10 ml ice-cold Wash Buffer, and carefully layered onto the sucrose gradients (10 ml mitochondrial suspension/gradient).


    Sucrose Step Gradient Purification of Mitochondria (Optional step)

    15. Make sucrose gradients consisting of 8 ml each of 30%, 52%, and 60% Sucrose Gradient Buffer layered from the top of tubes. The Sucrose Gradient Buffer is layered into 40 ml (e.g., SW 27 rotor) centrifuge tubes.

    16. Allow the gradients to equilibrate at 4oC overnight.
  • (or, may be made just prior to use if the 52 and 30% layers are added with sufficient rapidity to cause distortion at the interfaces. If the interfaces are too sharp, the mitochondria form a dense layer which may trap other organelles, resulting in a poorer separation.)


  • 17. Centrifuge the gradients for 60 minutes at 83,000g (e.g., 25,000 rpm in the SW 27 rotor) at 4oC.

    18. Carefully collect the mitochondria from the 30%|52% interface with sterile wide-bore pipettes.
  • (Electron microscopic examination shows that this fraction consists primarily of intact mitochondria, and is devoid of detectable plastids.)


  • 19. Dilute the mitochondria over a 15-minute period with three volumes of ice-cold Wash Buffer.
  • (Too rapid a dilution will cause osmotic shock and lysis of the organelles.)


  • 20. Collect the mitochondria by centrifugation at 8,500g for 20 minutes at 4oC in sterile test tubes, and resuspended in a small volume (1-5 ml) of ice-cold Wash Buffer.


    Lysis of Mitochondria and Isolation of Nucleic Acids

    21. Add ATA (RNase inhibitor) to make a final concentration of 1 mM to the mitochondrial solution.

    22. Lyse the mitochondria by adding 0.25 volume of Lysis Buffer. Mix gently.

    23. The lysate is extracted with an equal volume of acid phenol containing 0.1% 8-hydroxyquinoline and 50% (v/v) chloroform: IAA (24:1).

    24. Extract several times until there is a negligible interface.
  • (The presence of DNA may make the aqueous phase quite viscous and turbid.)


  • 25. Extract the aqueous phase once with chloroform: IAA (24:1, v/v).

    26. Precipitate the nucleic acids with 2.5 volumes of 95% ethanol at -20oC overnight.

    27. Centrifuge at 12,000g for 10 minutes to collect RNA.

  • (Alternatively, precipitate RNA by LiCl.

  • Add LiCl to a final concentration of 2 M.

  • Store the solution for at least 6 hours on ice prior to centrifugation.

  • Perform LiCl precipitation two times to ensure the purity of RNA.)


  • 28. Resuspend the RNA in 200-1000 ul of Storage Buffer depending on the expected yield.

    29. The optical density of a 1:100 dilution is read at 260, 280 and 340 nm to calculate the yield of RNA. Because ATA absorbs at 260 nm, it must be included in the blank cuvette at a concentration of 0.5 mM.

    29. Store RNA at -80oC.

    30. Analyze the intactness of RNA by northern analysis.




    NOTES

  • The sucrose step gradient can be scaled down using 12 ml SW 41 rotor or TST 41.14 rotor.


  • ATA inhibits polynucleotide kinase. Therefore, if the RNA is to be used for end-labeling, ATA should be omitted. The absence of a nuclease inhibitor may result in some degradation of the RNA, but its intactness is not germane to RNA labeling techniques that include partial alkaline hydrolysis. One may divide the mitochondria into aliquots to be lysed with or without ATA.


  • The advantage of using LiCl for RNA precipitation is that DNA is not precipitated. RNA species smaller than approximately 5.8 S will also remain in the supernatant. The DNA and smaller RNA species may be recovered by precipitation of the LiCl supernatant with ethanol.





  • KIT INFORMATION




    REFERENCES

  • Boutry, M., Faber, A.M., Charbonnier,M., and Briquet, M., 1984, Plant Mol. Biol. 3, 445.


  • Carmichael, G.G., 1980, Electrophoresis 1, 78.


  • Crews, S. and Attardi, G., 1980, Cell 19, 775.


  • Dyer, T.A. and Leaver, C.J., 1981, in "The Biochemistry of Plants" (P. K. Stumpf, E. Conn, and A. Marcus, eds.), p. III. Academic Press, New York.


  • Fox, T.D. and Leaver, C.J., 1981, Cell 26, 315.


  • Hackenbrock, C.R., 1968, J. Cell Biol. 37, 345.


  • Hallick, R.B., Chelm, B.K., Gray, R.W., and Orozco, E.M., 1977, Nucleic Acids Res. 4, 3055.


  • Kroon, M. and Saccone, C., eds., 1980, "The Organization and Expression of the Mitochondrial Genome." Elsevier/North-Holland Biomedical Press, Amsterdam.


  • Leaver, C.J. and Harmey, M.A., 1973, Biochem. Soc. Symp. 38, 175.


  • Lonsdale, D.M., Hodge, T.P., and Fauron, C.M.R., 1984, Nucleic Acids Res. 12, 9249.


  • Maizels, D., 1976, Cell 9, 431.


  • Moore, A.C. and Proudlove, M.O., 1983, in "The Biochemistry of Plants" (E. Conn and A. Marcus, eds.), p. 153. Academic Press, New York.


  • Nass, M.M.K. and Nass, S., 1963, J. Cell Biol. 19, 595-613.


  • Palmer, J.D. and Shields, C.R., 1984, Nature (London) 307, 5950.


  • Rigby, P.W.J., Dreckmann, M., Rhodes, C., and Berg, P., 1977, J. Mol. Biol. 113, 237.


  • Stern, S.B. and Palmer, D., 1984, Nucleic Acids Res. 12, 6141.


  • Stern, D.B. and Newton, K.J., 1985, Curr. Genet. 9, 395.


  • Thomas, B., 1980, P.N.A.S. 77, 5201.


  • Thompson, W.F., Everett, M., Polans, N.O., Jorgensen, R.A., and Palmer, J.D., 1983, Planta, 158, 487.



  • Please send your comment on this protocol to "editor@MolecularInfo.com".

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified