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