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In Situ Hybridization, Isotopic 35S-RIBOPROBE SYNTHESIS FOR ISOTOPIC In Situ HYBRIDIZATION
by Josiah N. Wilcox, Ph.D. (based on Melton et al., 1984)
  1. Pipet 12.5µl 35S-UTP (1200 Ci/mmol) into 1.5ml microfuge tube. Final concentration should be 12µM. Lyophilize in speed vac. (Do not use 35S-UTP that has been previously thawed. Order 250µCi vials and use up at first thawing or discard excess via radioactive disposal.)
  2. To tube with the dried probe add:

    2.0µl 5x Transcription buffer
    1.0µl DTT, 100mM
    1.0µl RNasin
    1.0µl DNA (linearized plasmid 1µg/µl)
    2.0µl GTP+CTP+ATP Mix (stock solution containing 2.5mM @)
    2.0µl Sterile dH2O

    Mix thoroughly and centrifuge.
    Add 1.0µl RNA Polymerase (SP6, T7 or T3 as required)
    Mix gently by pipeting, do not vortex, and incubate 1-2 hours, 37°C.
    (Order all of the above as ready-made stocks from Promega Biotech).

  3. To stop the reaction:

    Vortex
    Add 1.0µl RQ1 DNase to the transcription reaction above
    Incubate 15 min., 37°C.

    To extract RNA after DNase step, add to the reaction:
    20µl 1x TE
    1.0µl tRNA (50mg/ml)
    Vortex

  4. Equilibrate one QuickSpin G-50 Sephadex column (Boehringer Mannheim Catalog No. 100-616) to room temperature for each riboprobe (15-30 min. at room temperature).

    Invert column gently for 20-25 times to suspend the gel.

    Remove TOP cap first, followed by the bottom cap.

    Allow the fluid within the column to drip through by gravity.

    Cut a collection tube (comes with column) approx. 5mm from bottom, place column in the collection tube and place the assembly in a 15ml tube.

    Spin for 2 min at 1100g or 2500rpm (setting #6 on IEC centrifuge).

    Discard fluid and collection tube.

  5. Place the column in a new collection tube, add riboprobe to the CENTER of the column. Place the assembly gently into the 15ml tube and spin for 4 min at 1100g (#6 on the IEC). Remove the assembly gently with forceps, and measure the volume of riboprobe.
  6. To monitor incorporation:

    Take 1µl from reaction and place in microfuge tube.
    Add 99µl 1xTE.
    Pipet 1.0µl of 1/100 dilution onto a small piece of DE81 ion exchange paper
    Wash 3 times 5 min. in 0.5M NaPO4, pH7.4
    Wash 10 seconds in dH2O
    Rinse briefly (<10sec) in 100% etoh
    Dry throughly and count in 10ml scintillation fluid

  7. To the riboprobe add 1xTE to a final concentration of 300,000 CPM/µl. Use immediately for in situ hybridization or store 100µl aliquots at -70°;C up to one week in TE.
  8. Just prior to starting the hybridization step:

    Thaw the riboprobe and keep on ice.

  9. Make up Hybridization Mix as described in the in situ hybridization protocol