Molecular Techniques and Methods

Isolation of Poly A+-mRNA by
Oligo(dT)-Cellulose Chromatography

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION


The size of eukaryotic mRNAs are ranging from 0.3 kb to over 30 kb. The abundance of mRNAs is from fewer than 5 copies to over 30,000 copies per eukaryotic cell. Polyadenylated mRNAs can be separated from nonpolyadenylated transfer RNAs and ribosomal RNAs that account over 95% of total cellular RNA. The general principle is that an RNA-DNA hybrid is formed between the poly A+-tail and the oligo (dT)12-18-Cellulose at 400 mM Sodium chloride, and that excess rRNAs, tRNAs and other small RNAs can be washed away at this ionic strength. The major drawback of this procedure is the need to start with total RNA on a milligram scale to avoid substantial losses during chromatography, extraction with organics and precipitations.




MATERIALS AND SOLUTIONS

Regeneration and Preparation of Oligo(dT)-Cellulose
(1) Weigh 0.25 g oligo(dT)-cellulose in a 15 ml tube.
(2)Wash oligo(dT)-cellulose two times each with;
- 0.1 M NaOH ------------------------------- 10 ml
- DEPC-treated H2O ------------------------- 10 ml
- Elution buffer -------------------------------- 10 ml.
- Centrifuge for 5 minutes at 3,000 rpm each time to collect oligo(dT)-cellulose.
(3) Add 5 ml 2x Binding Buffer and 5 ml DEPC-treated H2O.
(4) Mix and equilibrate for 1-2 minutes.

  • For long term storage the matrix can be stored at 4oC in 1 X Binding Buffer + 0.02% Sodium azide (NaN3).



  • 2 X Binding Buffer (100 ml)
    20 mM Tris-HCl (pH 7.5) --------------------------- 2 ml of 1 M Tris-HCl
    800 mM NaCl --------------------------------------- 16 ml of 5 M NaCl
    2 mM EDTA ----------------------------------------- 0.4 ml of 0.5 M EDTA
    0.2% SDS -------------------------------------------- 2 ml of 10% SDS
    DEPC-treated H2O ---------------------------------- 79.6 ml


    Wash Buffer (100 ml)
    10 mM Tris-HCl (pH 7.5) --------------------------- 1 ml of 1 M Tris-HCl
    100 mM NaCl --------------------------------------- 2 ml of 5 M NaCl
    1 mM EDTA ---------------------------------------- 0.2 ml of 0.5 M EDTA
    0.1% SDS ------------------------------------------- 1 ml of 10% SDS
    DEPC-treated H2O ---------------------------------- 95.8 ml


    Elution Buffer (100 ml)
    10 mM Tris-HCl (pH 7.5) --------------------------- 1 ml of 1 M Tris-HCl
    1 mM EDTA ---------------------------------------- 0.2 ml of 0.5 M EDTA
    0.1% SDS ------------------------------------------- 1 ml of 10% SDS
    DEPC-treated H2O ---------------------------------- 97.8 ml


    DEPC-treated H2O

    Pasteur pipet, baked at 250oC overnight

    Glass wool, baked at 250oC overnight




    PROCEDURES

    1. Pour 0.25 g oligo(dT)-cellulose preparation into the pasteur pipette column with glass wool at the bottom.

    2. Equilibrate the column with 10 ml 1 X Binding Buffer.

    3. Dilute up to 50 mg total RNA sample to 250 ul with DEPC-treated H2O.

    4. Leave RNA sample at 65oC for 5 minutes.

    5. Add 250 ul 2 X Binding Buffer and apply the total RNA sample to the column.

    6. Collect the flow-through, leave it at 65oC for 5 minutes and reapply to the column.

    7. Wash the column with 5 ml 1 X Binding Buffer.

    8. Wash the column with 3 ml Wash buffer.

    9. Elute poly A+-mRNA with 3 ml Elution Buffer on ice.

    10. Add 0.1 vol of 3 M Sodium acetate (pH 7.0) and 2.5 vol of ice-cold absolute ethanol to precipitate poly A+-mRNA.

    11. An overnight precipitation at -20oC maximizes the precipitation of RNA.

    12. Centrifuge at 15,000g for 20 minutes to pellet the RNA.

    13. Redissolve the poly A+-mRNA pellet in 50 ul DEPC-treated H2O.

    14. Store RNA at -80oC.

    15. RNA is stable at -80oC for 6 months.

    16. Regenerate oligo(dT)-cellulose as in MATERIALS AND SOLUTIONS.




    NOTES

  • Maximum binding capacity ;
    50 mg total RNA / 0.25 g oligo(dT)-cellulose


  • SDS can be omitted from the 2 X Binding Buffer as it may precipitate in cold temperature and clog the column. Residual SDS may coprecipitate with the RNA and interfere with reverse transcription.





  • KIT INFORMATION

    Several kits are available, whereby poly-A+ RNA can be isolated directly from cell or tissue lysates on a small scale. Kits can be divided into two types:

  • Paramagnetic particles that either contain covalently attached oligo(dT) or streptavidin. The hybridization step is carried out in solution between poly-A+ RNA and biotin-tagged oligo(dT), and the hybrids are collected on the streptavidin-coated paramagnetic beads.


  • Small oligo(dT)-containing cellulose beads that are able to stay in a fine suspension during hybridization and is pelleted by centrifugation.





  • REFERENCES

  • Aviv, H, Leder, Pn (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. P.N.A.S. 69: 1408-1412.

  • Methods in Molecular Biology. vol. 86 (1998) RNA isolation and characterization protocols. Ralpley, R and Manning D.L. (eds.), Humana Press.



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

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