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  • Isolation of mRNA from Tissue: Using the Magnetic mRNA Isolation Kit.

    Overview

    Isolation Preparation

    Allow all kit components to come to room temperature.

    Resuspend Oligo d(T)25 beads by agitating at room temperature (RT) for 30 minutes.

    Reuse Oligo d(T)25 beads for second-round poly(A)+ selection.

    Oligo d(T)25 beads can be re-used for a second-round of purification of a poly(A)+ RNA eluent without regeneration. Wash beads once with an additional 100 μl of elution buffer. Place in magnetic rack and pull beads to the side of the tube. Remove and discard wash solution. Wash beads once with 200 μl of Lysis/Binding Buffer then re-apply previously isolated eluent to beads after adjusting salt concentration to 0.5 M LiCl or NaCl. Repeat isolation steps.

    Introduction

    Regeneration of Oligo d(T)25 beads.

    Oligo d(T)25 beads can be regenerated and used to isolate RNA from a different source. Add 0.1 NaOH to the beads and incubate at room temperature with agitation for 5 minutes. Wash beads twice with sterile RNase-free 1X PBS (pH 7.4) containing 0.1% Tween 20. Then store in the same buffer.

    Precautions should be taken to avoid ribonuclease contamination during isolation of the RNA.  All materials used during the isolation procedure should be sterile and RNase-free.

    Clean the homogenizer with a NaOH based detergent and rinse with sterile RNase-free deionized H2O.

    Wear latex gloves or equivalent at all times when handling kit components.

    Reserve reagents exclusively for RNA work. If possible, separate procedures such as plasmid preps which require the use of RNases from this work area.

    Increasing the amount of tissue sample beyond the recommended amount at a given scale does not increase the yield of poly(A)+ RNA isolated. More importantly, this will cause an increase in the amount of tissue debris and ribonucleases present during the isolation.

    Protocol

    1. Aliquot the appropriate volume of Oligo d(T)25 beads for the scale of isolation (Table 2).
    2. Add 200 μl of Lysis/Binding Buffer to beads, vortex briefly and mix with agitation for 2 minutes. Beads should remain in the lysis/binding wash solution until removal immediately before adding the cell lysate.
    3. If tissue has been prepared beforehand and frozen, add Lysis/Binding directly to frozen tissue sample and proceed to Step 4, below.
    4. Weigh fresh or frozen animal itssue as quickly as possible to avoid mRNA degradation.
    5. Place tissue into liquid nitrogen and grind immediately.
    6. Transfer ground tissue to the Lysis/Binding Buffer and homogenize using brief 20-second pulses.
    7. If the solution is viscous, pass the lysate through a 21-gauge needle attached to a 1 or 2 ml syringe. A noticeable decrease in viscosity should be observed.
    8. Spin sheared lysate at 12,000 rpm, 4oC for 1 minute.
    9. Place the microcentrifuge tube containing the beads and lysis/binding wash into the magnetic rack and pull the magnetic beads to the side of the tube, remove and discard the wash solution.
    10. Decant tissue supernatant and add to previously washed Oligo d(T)25 beads. Place lysate-and-bead suspension on the agitator and incubate at RT for 10 minutes.
    11. Place microcentrifuge tube into the magnetic rack and pull magnetic beads to the side of the tube, remove and discard supernatant.
    12. Add the appropriate volume of Wash Buffer 1 (Table 2) to the beads, vortex gently to suspend beads. Incubate with agitation for 1 minute.
    13. Place microcentrifuge tube in the magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution.
    14. Repeat once.
    15. Add the appropriate volume of Wash Buffer 2 (Table 2) to the beads and mix with agitation for 1 minute.
    16. Place microcentrifuge tube in the magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution.
    17. Repeat once.
    18. Add the appropriate volume of Low Salt Buffer (Table 2) to the beads and mix with agitation for 1 minute.
    19. Place tube in magnetic rack and pull magnetic beads to the side of the tube, remove and discard wash solution. 
    20. Add 100 μl of Elution buffer and vortex gently to suspend beads.
    21. Incubate at 50oC for 2 minutes with occasional agitation to elute poly(A)+ RNA.  >90% of the poly(A)+ RNA bound to the beads is recovered in this step.
    22. Place microcentrifuge tube in the magnetic rack and pull magnetic beads to the side of the tube.
    23. Transfer eluent to a clean, sterile RNase-free tube. Store on ice and immediately quantitate of place at -80oC for long-term storage.