Chuck Perou’s mRNA labeling Protocol

 

  1. RT Setup

    2. Anchored Oligo dT primer----2 microliters----(2.5 micrograms/microliter)

    Controls (if used)--------------2 microliters

    mRNA -------------------------(what ever is needed to reach 1.5-2 micrograms)

    DEPC/H2O--------------------up to 16 microliters

    FINAL VOLUME EQUALS 16 MICROLITERS

  2. Heat at 70 C for 10 minutes
  3. Chill on ice for 1-2 minutes
  4. RT Reaction for each individual tube

    5. 5X RT Buffer-------6 microliters

    50X dNTPs---------0.7 microliters-----(500mm A,C,G, 200mm T)

    Cy Dyes dUTP-----3 microliters--------(either Cy3 or Cy5)

    DTT Stock----------3 microliters--------(comes with RT setup)

    Superscript II RT--1.7 microliters------(cat# 18064-014 Gibco-BRL)

  5. Mix well
  6. 42 C for 1 hour
  7. Add another 1 microliter of Superscript II RT and mix
  8. 42 C for 1 more hour
  9. Degrade mRNA with 1.5 microliters of 1M NaOH / 2mM EDTA
  10. 65 C for 8 minutes (do NOT go TOO long here)
  11. add 15 microliters of 0.1M HCL
  12. Add 450 microliters of T.E (7.4) to each sample and place each sample into a microcon-30 filter
  13. Add 15 microliters of Human COT1 DNA (Gibco-BRL = 1 microgram/microliter) to each sample in the microcon filter
  14. Spin in Eppendorf centrifuge until volume equals about 50 microliters (8-10')
  15. Remove flow throughs, and pool Cy3 and Cy5 flow throughs together for future recover of Cy dyes (store at -20 C).
  16. Invert microcons, recover labeled samples, and pool Cy3 and Cy5 samples together that will be used for an individual experiment, in a single microcon filter that was used in step 15
  17. Add 500 microliters of T.E again, and spin until final volume equals 8 microliters or less (BE VERY CAREFUL TO NOT SPIN THE SAMPLE DRY!!!)
  18. To the 8 microliter combined Cy3 + Cy5 sample, add the following

    19. Yeast tRNA--------1 microliter-----(10 micrograms/microliter)

    PolyA DNA-------2 microliters-----(10 micrograms/microliter)

    20XSSC-----------2 microliters-----(FINAL SSC concentration approximately 3X)

    10% SDS----------0.3 microliters

    FINAL VOLUME IS = 13.3 MICROLITERS

  19. Mix Well
  20. Heat sample at 100 C for 2 minutes, spin very briefly
  21. Place samples at 42 C for 20-30 minutes
  22. During Step 21, get and set up the necessary number of Hyb chambers and get 22mm X 22mm coverslips ready, and get arrays ready
  23. Add the 13 microliters of probe onto the center of the array while NOT actually touching the array face with the pipette tip.
  24. Quickly and gently place the 22mm X 22mm glass#1 coverslip onto the array face.
  25. Add about 15-20 microliters of 3XSSC in two drops onto the end of the array slide away from the actual array for hydration purposes.
  26. Assemble the Hyb chamber with the array slide in it, and place into a 65 C water bath overnight.
  27. Pull out the hyb chamber and dry off the excess H2O
  28. Disassemble the hyb chamber, and quickly place the slides into a slide washing chamber that contains 2XSSC/0.05%SDS, and giggle the slide holder up and down until the slide coverslip falls off. Repeat this individually for each array, one at a time, until all are done
  29. Wash slides in 1XSSC for 3-5 minutes
  30. Wash slides in 50 C 0.2XSSC for 3-5 minutes, twice
  31. Spin slides down in centrifuge at 200 RPM for 2 minutes
  32. SCAN immediately