Silver: RNA Export Assay

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(adapted fr. E. Lei, Feb. 2002)

  1. Grow 5mL of culture to early log phase (1 x 107 to 2 x 107 cells/mL).
    • Temperature shift if desired – use waterbath for shorter temperature shift or warm room for longer (4h or so)
  2. Fix by adding culture to 15mL Falcon tube containing 350µL 37% formaldehyde (inside the chemical hood, dispose the waste in the proper waste jug inside the hood!).
    • Mix well.
  3. Incubate 1 hr with agitation at the temperature at which the cells were grown.
    • For zero timepoints, I sometimes fix cells at the high temperature.
  4. Spin down cells for 2 min at 2000 x g.
  5. Wash twice with 1mL 0.1M phosphate buffer (pH 6.5)
  6. Flash spin to pellet cells and discard sup, followed by a single wash with 1mL P sol’n.
  7. Discard waste via aspiration.
  8. Resuspend pellet in 100µL to 1mL P sol’n to desired cell density. (A culture at 1 x 107 cells/mL can be resuspended in 200µL)
    • Store at 4˚C if desired. Cells are good for months if not contaminated. THINK OF YOUR CONTROLS: P soln only, untreated cells, cells w/o hyb by probes, your basic (+) & (-) controls (i.e. export mutant & WT), etc.
  9. Add 1µL 1M DTT to 100µL cells and incubate at RT for 10 min.
  10. Add 3µL 10mg/mL zymolyase (-20°C storage, 50µl aliquots).
    • The average strain requires a 5 min incubation at RT, put on ice immediately! Temperature-shifted cells are often more resistant to digestion. Do a single digestion to get a sense of how much time it requires for your desired digestion – then do all the digestions in the set of 6 samples!
  11. Check your digestion under microscope by adding 2µL of digested cells to 1µL 0.3%SDS on a microslide. Add 2 µl of undigested cells & 1 µl of 0.3% SDS for comparison purpose.
  12. Look for % of dark/halo cells. Aim for 10-50% lysis (dark cells). Do all steps on ice from this point on.
  13. Spin down cells briefly and resuspend in 100µL P sol’n, leave on ice.
  14. Coat slide wells with 0.3% polylysine (-20°C storage) – just a bubble with your P200 pipetteperson.
  15. Incubate for 15 min at RT inside a humidified chamber (a P1000 box with foil coating the cover and fill the box 1/2 way with water).
  16. Remove the polylysine by aspiration (gently from the edge, do not touch the slide).
  17. Rinse slide once with water and air dry.
  18. Apply 25µL cell suspension to each well; Incubate 10min at RT; Remove cell suspension by aspiration as above.
    • Do not let wells dry out from this point on. If in a pinch, leave cells in P sol’n.
  19. Permeabilize cells on the slide with 0.5% Triton X-100 in P sol’n. (5µL TX100 + 1mL P sol’n) - just a small drop of bubble on the slide! Treat for 10min. Remove suspension with aspiration as above.
  20. While permeabilizing, make 0.1M triethanolamine (TEA) pH 8.0 (1.85g in 100mL water, pH with NaOH (approximately 2-5 ml with 10N soln).
    • FRESH EACH TIME!!
  21. Rinse cells on the slide with P sol’n, a bubble as before, remove as before.
  22. Equilibrate cells on the slide with freshly prepared 0.1M TEA for 2min at RT.
  23. Block polar groups with 0.25% acetic anhydride (under the hood in the chemical cabinet, do this in the flow hood) in 0.1M TEA (2.5µL in 1mL) for 10min at RT. Remove as before.
  24. Add prehyb (stored at 4°C) + tRNA (yeast) to each well. Prehyb 1h at 37˚C in a humidified chamber.
  25. Add Cy3-dT50 probe (stored at –20°C) to prehyb + tRNA at 1:200 (up to 1:500). Vortex well.
  26. Add prehyb + probe to wells. Hybridize O/N at 37˚C in a humidified chamber.
  27. Remove probe/prehyb suspension via aspiration. Wash cells on the slide once quickly with 2 x SSC at RT
  28. Do the successive wash steps as follows, a bubble each time and aspirate at the end of each wash:
    1. 1h in 2 x SSC at RT
    2. 1h in 1 x SSC at RT
    3. 30min in 0.5 x SSC at 37˚C
    4. 30min in 0.5 x SSC at RT
  29. Stain nuclei with 10µg/mL (dilute the lab stock in –20°C accordingly) DAPI in 1xPBS for 1min at RT (need about 25µl per sample).
  30. Wash cells 3x quickly with 1xPBS.
  31. Air dry slide completely and mount the slide using the mounting media (a bubble on each well, the lab stock is at 4°C).
    • Gently lay down the cover slip from an angle (size varies based on number of samples and slides used). Make sure no bubble is trapped in the well. Remove excess mounting agent from the edges with kimpwipes. Seal the slide edges with clear nail polish.
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