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  • Isolate mRNA using the NEBNext Oligo d(T)25 Magnetic Beads (E7490)

    Starting Material: 1–5 µg of total RNA.

    1. Dilute the total RNA with nuclease-free water to a final volume of 50 μl in a nuclease-free 0.2 ml PCR tube. 

    2. Aliquot 15 μl of NEBNext Magnetic Oligo d(T)25 Beads into nuclease-free 0.2 ml PCR tube. 

    3. Wash the beads two times with 100 μl of RNA Binding Buffer and remove the supernatant. 

    4. Resuspend the beads in 50 μl of RNA Binding Buffer and add the 50 μl of total RNA sample from step 1.

    5. Place the tubes on the thermal cycler and heat the sample at 65°C for 5 minutes and hold at 4°C to denature the RNA and facilitate binding of the poly-A-RNA to the beads. 

    6. Remove tubes from the thermal cycler when the temperature reaches 4°C. 

    7. Place the tubes on the bench and incubate at room temperature for 5 minutes to allow the RNA to bind to the beads.

    8. Place the tubes on the magnetic rack at room temperature for 2 minutes to separate the poly-A RNA bound to the beads from the solution.

    9. Remove and discard all of the supernatant. Take care not to disturb the beads. 

    10. Remove the plate from the magnetic rack. 

    11. Wash the beads twice with 200 μl of Wash Buffer to remove unbound RNA. Pipette the entire volume up and down 6 times to mix thoroughly.

    12. Place the tubes on the magnetic rack at room temperature for 2 minutes. 

    13. Remove and discard all the supernatant from each well of the plate using a multichannel pipette. Take care not to disturb the beads.

    14. Remove the tubes from the magnetic rack.

    15. Add 50 μl of Tris buffer to each well of the plate. Gently pipette the entire volume up and down 6 times to mix thoroughly. 

    16. Place the tubes on the thermal cycler. Close the lid and heat the sample at 80°C for 2 minutes, then hold at 25°C to elute the poly-A RNA from the beads. 

    17. Remove the tubes from the thermal cycler when the temperature reaches 25°C.

    18. Add 50 μl of RNA binding buffer to each sample to allow the RNA to bind to the beads. Gently pipette the entire volume up and down 6 times to mix thoroughly.

    19. Incubate the tubes at room temperature for 5 minutes. 

    20. Place the tubes on the magnetic stand at room temperature for 2 minutes. 

    21. Remove and discard all of the supernatant from each tube. Take care not to disturb the beads. 

    22. Remove the tubes from the magnetic stand. 

    23. Wash the beads twice with 200 μl of Wash Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly.

    24. Place the tubes on the magnetic stand at room temperature for 2 minutes. 

    25. Remove and discard all of the supernatant from each tube. Take care not to disturb the beads. 

    26. Remove the tubes from the magnetic stand.

    27. Elute the mRNA from the beads by adding 17 µl of the Tris Buffer and incubating the sample at 80°C for 2 minutes. Immediately, place the tubes on the magnetic rack. 

    28. Collect the purified mRNA by transferring the supernatant to a clean nuclease-free PCR Tube. 

    29. Place tube on ice. 

    30. Assess the Yield and the Size Distribution of the purified mRNA. Run 1 µl on the Bioanalyzer® (Agilent Technologies, Inc.) using a RNA Pico Chip.