RNA Detection

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  1. Pass the lysate with a syringe several times through a 23- and
    26-G needle (seeNote 4). Afterward, save 0.2 mL of input
    lysate for downstream analyses (e.g., Western analysis and RNA
    extraction) and store it at 80 C.

  2. Pre-wash the magnetic oligo(dT) beads in 5 mL of lysis/bind-
    ing buffer in a 15 mL Falcon tube and concentrate them on a
    magnet. Afterward, resuspend beads into the cell extract by
    first taking them up in 1 mL of the prepared lysate and trans-
    ferring them to the tube containing the remaining lysate (see
    Note 5).

  3. Incubate for 1 h at room temperature on a rotating wheel (see
    Note 6).

  4. Concentrate beads on magnet. The initial concentration of the
    beads on the magnet may take from 10 min to 1 h.

  5. Carefully collect supernatant and place it into a new tube for
    additional rounds of oligo(dT) precipitation (seeNote 7).

  6. Wash beads two times in one sample volume of room-
    temperature lysis/binding buffer. For each washing step, rotate
    the tubes 2–5 min on a rotating wheel at room temperature.
    Afterward, concentrate the beads on magnet and carefully
    remove the supernatant (seeNote 8).

  7. Wash beads two times in one sample volume of room-
    temperature washing buffer containing 0.5 mM DTT and
    complete EDTA-free protease inhibitor cocktail. For each
    washing step, rotate the sample 2–5 min on a rotating wheel
    at room temperature. Afterward, concentrate the beads on
    magnet and carefully remove the supernatant.

  8. For the third washing step, resuspend beads in 1 mL of washing
    buffer and transfer to a fresh 1.5-mL tube. Afterward, concen-
    trate the beads on magnet and remove the supernatant.

  9. Wash beads once with 1 mL of ice-cold low-salt elution buffer
    to remove traces of IGEPAL CA-630 (seeNote 9). Concen-
    trate beads with magnet and remove supernatant.

  10. Resuspend beads in 0.5–1 mL of low-salt elution buffer and
    incubate them for 2 min at 80C. Remove supernatant into a
    new precooled 15-mL falcon tube and place it on ice as quickly
    as possible. Pool the eluates from different aliquots. Once the
    eluate is removed, immediately cool down the beads by placing
    them on ice (seeNote 10).

  11. Repeat the elutionstep 11once more.

  12. Perform several additional rounds of oligo(dT) affinity purifi-
    cations. We typically perform two additional rounds with only
    one elution per round for a total of three affinity purifications
    (seeNote 11).


410 Miha Milek and Markus Landthaler

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