Molecular Techniques and Methods

Isolation of RNA for mRNA Differential Display

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Phosphate-Buffered Saline (PBS)


Extraction Buffer (100 ml)
0.14 M NaCl --------------------------------------- 2.8 ml of 5 M NaCl
10 mM Tris-HCl (pH 7.5) -------------------------- 1 ml of 1 M Tris-HCl
1.5 mM MgCl2 ------------------------------------- 0.15 ml of 1 M MgCl2
DEPC-treated H2O -------------------------------- 96.05 ml


Lysis Solution
Guanidinium thiocyanate ---------------------------- 100 g
Distilled H2O --------------------------------------- 117 ml
Sodium citrate (pH 7.0) ----------------------------- 5.25 ml of 1 M Sodium citrate
10% Sarkosyl -------------------------------------- 10.5 ml
  • Dissolve at 65oC, and store at room temperature for several months.
  • Before use, add 72 ul of 2-mercaptoethanol to 10 ml of lysis solution.




    PROCEDURES

    1. Wash 4 x 106 cells with PBS.

    2. Gently resuspend cells in 450 ul of Extraction Buffer.

    3. Dropwise, add 50 ul of 5% Nonidet NP-40 and mix.

    4. Leave on ice for 2 minutes.

    6. Spin for 5 minutes at 550g and 4oC.

    7. Transfer the supernatant into a fresh tube containing 4.5 ml of Lysis Solution and mix.

    8. Leave 10 minutes on ice.

    9. Add 450 ul of 2 M Sodium acetate (pH 5), and 4.5 ml of acid phenol.

    10. Shake for 5 minutes.

    11. Add 1.5 ml chloroform: isoamyl alcohol (49:1).

    12. Spin at 4,000g for 10 minutes.

    13. Take the upper phase, add 5.7 ml of isopropanol, and leave on ice for 20 minutes.

    14. Spin at 4,500 rpm for 30 minutes.

    15. Resuspend the pellet in 0.3 ml of Lysis Solution, transfer to a microfuge tube, and incubate on ice for 15 minutes.

    16. Precipitate the RNA with 1 ml of 96% ethanol at -70oC.

    17. Spin at full speed for 10 minutes, wash the pellet with 1 ml 70% ethanol, and spin down again.

    18. Remove the supernatant and resuspend the pellet in 200 ul of DEPC-treated H2O.

    19. Add the followings for DNase (RNase-free) reaction.
    1 M Tris-HCl (pH 7.5) ---------------------- 10 ul
    1 M MgCl2 ---------------------------------- 2 ul
    RNase-free DNase -------------------------- 2 ul
    RNase Inhibitor ------------------------------ 0.5 ul

    20. Incubate for 15 minutes at 37oC.

    21. Add 200 ul of phenol: chloroform (1:1), vortex, and spin to separate the two phases.

    22. Take the supernatant and add 20 ul of 2 M Sodium acetate (pH 4.0), and 1 ml 96% ethanol. Cool down to -70oC for 10 minutes.

    23. Spin for 10 minutes, and wash the pellet with 70% ethanol.

    24. Resuspend the pellet in 10-20 ul of DEPC-treated H2O and measure the concentration.

    25. RNA should be stored in 100% ethanol at -70oC.




    NOTES




    KIT INFORMATION




    REFERENCES


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

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