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  • Protocol for Use with Total RNA (E7525)

    Symbols

    This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input.

    Protocol

    Starting Material: Total RNA (10 ng–1 μg) purified mRNA (10–100 ng), or ribosomal depleted total RNA (10–100 ng)

    Note: Follow steps in Protocol (A) if starting material is total RNA. Perform mRNA isolation, fragmentation and priming using the NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB #E7490). If starting material is purified mRNA or ribosomal depleted RNA, proceed to (B).

    (A) Preparation of First Strand Reaction Buffer and Random Primer Mix

    Prepare the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) as follows in a nuclease-free tube:
    NEBNext First Strand Synthesis Reaction Buffer (5X)   8 μl
    NEBNext Random Primers   2 μl
    Nuclease-free water   10 μl
    ---------------------------------------------------
    Total Volume   20 μl

    Note: Keep the mix on ice during the mRNA isolation.

    mRNA Isolation, Fragmentation and Priming Starting with 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 and keep on ice.
    2. Aliquot 15 μl of NEBNext Oligo d(T)25 beads into a nuclease-free 0.2 ml PCR tube.
    3.  Wash the beads by adding 100 μl of 2X RNA Binding Buffer to the beads. Pipette the entire volume up and down 6 times to mix thoroughly.
    4. Place the tubes on the magnetic rack at room temperature for 2 minutes.
    5. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.
    6. Remove the tube from the magnetic rack.
    7. Repeat steps 3–6.
    8. Resuspend the beads in 50 μl of 2X RNA binding Buffer and add the 50 μl of total RNA sample from step 1.
    9. Place the tube in a thermocycler and close the lid. 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 mRNA to the beads.
    10. Remove the tube from the thermocycler when the temperature reaches 4°C.
    11. Place the tube on the bench and incubate at room temperature for 5 minutes to allow the mRNA to bind to the beads.
    12. Place the tube on the magnetic rack at room temperature for 2 minutes to separate the poly-A mRNA bound to the beads from the solution.
    13. Remove and discard all of the supernatant. Take care not to disturb the beads.
    14. Remove the tube from the magnetic rack.
    15. Wash the beads by adding 200 μl of Wash Buffer to the tube to remove unbound RNA. Pipette the entire volume up and down 6 times to mix thoroughly.
    16. Place the tube on the magnetic rack at room temperature for 2 minutes.
    17. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.
    18. Remove the tube from the magnetic rack.
    19. Repeat steps 15–18.
    20. Add 50 μl of elution buffer to each tube. Gently pipette the entire volume up and down 6 times to mix thoroughly.
    21. Place the tube in the thermocycler. Close the lid and heat the samples at 80°C for 2 minutes, then hold at 25°C to elute the Poly-A mRNA from the beads.
    22. Remove the tube from the thermocycler when the temperature reaches 25°C.
    23. Add 50 μl of 2X RNA Binding Buffer to the sample to allow the mRNA to re-bind to the beads. Gently pipette the entire volume up and down 6 times to mix thoroughly.
    24. Incubate the tube at room temperature for 5 minutes.
    25. Place the tube on the magnetic rack at room temperature for 2 minutes.
    26. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.
    27. Remove the tube from the magnetic rack.
    28. Wash the beads by adding 200 μl of Wash Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly.
    29. Place the tube on the magnetic rack at room temperature for 2 minutes.
    30. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.
    31. Remove the tubes from the magnetic rack.
    32. Repeat steps 28–31.
    33. Wash the beads by adding 200 μl of Elution Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly.
    34. Place the tube on the magnetic rack at room temperature for 2 minutes.
    35. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.
      Note: It is important to remove all of the supernatant to successfully fragment the mRNA in the subsequent steps.
    36. Remove the tube from the magnetic rack.
    37. Elute mRNA from the beads by adding 15 μl of the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) prepared at the start of the protocol (pages 2–3) and incubating the sample at 94°C for 15 minutes. Immediately, place the tubes on the magnetic rack.
    38. Collect the purified mRNA by transferring the 10 μl of supernatant to a clean nuclease-free PCR Tube.
    39. Place the tube on ice.
    40. Proceed to First Strand cDNA Synthesis:
    (B) RNA Fragmentation and Priming Starting from Purified mRNA or ribosomal depleted mRNA:

    Purified mRNA/ribosomal depleted RNA (10–100 ng)   5 μl
    NEBNext First Strand Synthesis Reaction Buffer (5X)   4 μl
    Random Primers   1 μl
    --------------------------------------------------------
    Final volume   10 μl
    1. Incubate the sample at 94°C for 15 minutes.
    2. Transfer the tube to ice.
    3. Proceed to First Strand cDNA Synthesis.

    First Strand cDNA Synthesis

    Note: If you are performing first strand synthesis as part of a directional RNA sequence workflow, it is recommended to add Actinomycin D to the reaction.

    1.  Dilute Actinomycin D stock solution (5 μg/μl) to 0.1 μg/μl in nuclease free water for immediate use.

      Note: Dilute solutions of Actinomycin D are very sensitive to light. In solution, Actinomycin D tends to adsorb to plastic and glass. For these reasons, unused dilute solutions should be discarded and not stored for further use. However, frozen aliquots of a concentrated stock solution (5 μg/μl) are expected to be stable for at least a month at -20°C.

      To the fragmented and primed mRNA (10 μl from section A step 39 or section B step 2) add the following components:

      (A) Non-Directional Reaction Step Up:
      Murine RNase Inhibitor   0.5 μl
      ProtoScript II Reverse Transcriptase   1 μl
      Nuclease free water   8.5 μl
      --------------------------------------------------------------------
      Final volume   20 μl

      (B) Directional Reaction Step Up:
      Murine RNase Inhibitor   0.5 μl
      Actinomycin D (0.1 μg/μl)   5 μl
      ProtoScript II Reverse Transcriptase   1 μl
      Nuclease free water   3.5 μl
      --------------------------------------------------------------------
      Final volume   20 μl

    2. Incubate the sample in a preheated thermocycler as follows:
      10 minutes at 25°C
      50 minutes at 42°C
      15 minutes at 70°C
      Hold at 4°C

    3. Immediately, perform second strand synthesis reaction, using NEBNext mRNA Second Strand Synthesis Module (NEB #E6111), or NEBNext Ultra Directional RNA Second Strand Synthesis Module (NEB #E7550).