Molecular Techniques and Methods

Isolation of Polysomal RNA from Plant Tissues

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Most plant tissue contains high levels of ribonuclease activity in the vacuoles. During the RNA extraction, RNA should be protected against this endogenous ribonuclease. In this procedure, alkaline pH of the Polysome Buffer and the presence of a chelating agent (EGTA) are used to prevent RNA degradation.

The RNA obtained was shown to be suitable for northern hybridization, in vitro translation, and cDNA synthesis. The 2-D electrophoretic pattern of in vitro translation products of polysomal RNA obtained this way was fully comparable to that of total RNA from the same tissue, indicating that no loss of specific sequences had occurred.

Polysomal RNA will be used in experiments that require the exclusive presence of actively translated mRNAs.




MATERIALS AND SOLUTIONS

Polysome Buffer (100 ml)
1 % Nonidet P40 --------------------------------- 2 ml of 50% Nonidet P40
50 mM MgCl2 ------------------------------------ 5 ml of 1 M MgCl2
25 mM EGTA ------------------------------------ 5 ml of 0.5 M EGTA
50 mM Tris-HCl (pH 9.0) ------------------------ 5 ml of 1 M Tris-HCl
250 mM NaCl ------------------------------------ 5 ml of 5 M NaCl
DEPC-treated H2O ------------------------------- 78 ml
  • Store at 4oC up to 1 month.


    Gradient Buffer (100 ml)
    10 mM MgCl2 ------------------------------------ 1 ml of 1 M MgCl2
    5 mM EGTA ------------------------------------- 1 ml of 0.5 M EGTA
    10 mM Tris-HCl (pH 8.5) ------------------------ 1 ml of 1 M Tris-HCl
    50 mM NaCl ------------------------------------- 1 ml of 5 M NaCl
    DEPC-treated H2O ------------------------------- 96 ml
  • Storage at 4oC up to1 month.


    Gradient Buffer + 0.2 M EDTA


    75% (w/v) Sucrose in Gradient Buffer
    Sucrose ------------------------------------------ 590 g
    Gradient Buffer ---------------------------------- 410 ml
  • Treat 0.1 % (v/v) of DEPC, stir slowly overnight and heat at 60oC for 1 hour. Store at 4oC up to 2 months.


    60%, 40%, 25%, 10% (w/v) Sucrose in Gradient Buffer
    Dilute 75% (w/v) Sucrose in Gradient Buffer by Gradient Buffer.


    Acid Phenol (pH 4.5)




    PROCEDURES

    1. Grind 5 g of plant tissues in liquid nitrogen in a mortar and pestle.

    2. Transfer the frozen powder to a beaker containing 20 ml ice-cold Polysome Buffer. Gently suspend the powder.

    3. Centrifuge for 10 min at 0oC and 27,000g.

    4. Filter the supernatant solution through a 3G-1 baked glass filter into a mass cylinder in ice.

    5. Transfer filtrate to Beckmann polycarbonate tubes (type 65 or type 42.1) and fill to approximately 80%.

    6. Underlayer the filtrate with 2 ml (Type 65) [or, 5 ml (Type 42.1)] of 60% (w/v) Sucrose in Gradient Buffer with a pasteur pipette

    7. Fill up tubes with filtrate.

    8. Centrifuge for 2 hours (Type 65) at 75,000g and 0oC [or, 3 hours (Type 42.1) at 65,000g].

    9. Decant supernatants carefully and place the tubes inverted on sterile tissues to drain off remaining liquid from the opalescent polysome pellets. The pellets can either be resuspended directly or be quickly frozen in liquid nitrogen and stored at -80oC.

    10. Prepare linear 10-40% (w/v) Sucrose gradients in Gradient Buffer in polyallomer SW28 or SW40 tubes (Beckmann) and keep in ice.

    11. Carefully resuspend the polysome pellets by pipetting in Gradient Buffer to a final concentration of approximately 2.5 mg/ml of polysomal RNA (A260=60 units), which is about 5 mg/ml of polysomes.

    12. Carefully load 20 mg of polysomes on SW28 gradient (or, up to 5 mg of polysomes on SW40 gradient).

    13. Centrifuge for 30 min at 0oC and 65,000g (SW40Ti) without a brake or 70 min at 45,000g (SW28) is sufficient to yield clear separation between residual monosomes and most of the contaminating hnRNA and polysomes > 100 S. Usually it is sufficient to monitor completely only 1 out of 6 gradients by A280 or A260 extinction in the flowcell and to fractionate the remainder after identification of the monosome peak and polysomes >100 S.

    14. Polysome-containing fractions are now collected and precipitated overnight at -20oC by addition of 1/10th volume of 3 M Sodium acetate (pH 7.0) and 2 volumes of 100% ethanol.

    15. The precipitate is collected by centrifugation at 20,000g for 30 min at 4oC, washed once with 70% ethanol and dried under vacuum.

    16. The precipitated polysomes can be extracted to yield polysomal RNA or can be EDTA-released to remove residual traces of hnRNA.

    For EDTA release, dissolve the dried pellets in Gradient Buffer with 0.2 M EDTA to a concentration of approximately 5 mg/ml of polysomes, incubate at 25oC for 30 minutes and recentrifuge on a linear 10-40% sucrose gradient. Protein-RNA complexes now sedimenting at the position of the monosomes are recovered as above by ethanol precipitation. This fraction contains the mRNAs, which can be purified by oligo(dT) cellulose chromatography.

    17. Before RNA extraction the polysomes or monosomes are first dissolved to approximately 1 mg/ ml. Extract RNA from ethanol-precipitated polysomes or monosomes by equal volume of acid phenol: chloroform: IAA.

    18. Add 140 ul 10 M LiCl per 500 ul RNA solution. Precipitate RNA at 4oC for 3 hours.

    19. Centrifuge at 10,000g for 15 minutes at 4oC.

    20. Resuspend the RNA pellet in 300 ul 1 mM EDTA.

    21.Check the RNA quality.




    NOTES

  • Centrifugation times should be adjusted to recover all the polysomes >100 S while leaving the monosomes on top of the sucrose cushion. The centrifugation times needed should be checked before large-scale preparation. Do not use a brake in centrifugation step. The rotor g values over 450,000 may severely damage the polysomes.


  • EDTA release is applied only when even very low levels of contamination with hnRNA must be prevented as is the case in single-copy DNA/mRNA saturation hybridizations.





  • KIT INFORMATION




    REFERENCES

  • Davies, E, Larkins, BA, Knight, RH (1972) Polyribosomes from peas: An improved method for their isolation in the absence of ribonuclease inhibitors. Plant Physiol. 50: 581-584.


  • Govers, F, Gloudemans, T, Moerman, M, Van Kammen, A, Bisseling, T (1985) Expression of plant genes during the development of pea root modules. EMBO 14: 861-867.


  • Gray, T, Cashmore, AR (1976) RNA synthesis in plant leaf tissue: The characterization of messenger RNA species lacking and containing polyadenylic acid. J. Mol. Biol. 108: 595-608.


  • Hari, V (1980) Polysomes from expanded tobacco leaves. Planta 148: 491-497.


  • Heinze, H, Herzfeld, F, Kuiper, M (1980) Light-induced appearance of polysomal poly(A)-rich messenger RNA during greening of barley plants. Eur. J. Biochem. 111: 137-144.


  • Hoge. JHC, Springer, J, Wessels, JGH (1982) Changes in complex RNA during fruit-body initiation in the fungus Schizophylium commune. Exp. Mycol. 6: 233-243.


  • Jackson, AD, Larkins, BA (1976) Influence of ionic strength, pH, and chelation of divalent metals on isolation of polyribosomes from tobacco leaves. Plant Physiol. 57: 5-10.


  • Loening, UE (1967) The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102: 251-257.


  • Sambrook, J, Fritsch, EF, Maniatis, T (1988) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.



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