Hybridization and Making RNA Probes

Descriptions of all solutions can be found in Reagents and Recipes.

Hybridization of embryos with labeled RNA probes

  1. Gently distribute ~50 µl pre-hybridized embryos per 1.5 ml eppendorf tube.
  2. Prepare the hybridization solution containing the labeled probes. Dilute the probe stocks in 100 µl hybridization solution per tube of embryos. Use 0.5-2.0 µl per probe per 100 µl hybridization solution, adjusting the amount according to the previously observed signal-to-noise ratio on embryos.
  3. Heat the probe mixture to 80°-85° C. for 2-3 minutes, then chill on ice. This step will denature any secondary structure that may have formed in the RNA.
  4. Drain as much of the pre-hybridization solution off the embryos as possible, tapping the tube to make them settle completely, then add the probe mixture to the embryos and put the tube back in the 55° C. water bath.
  5. Stir the embryos with a few finger taps on the tube after adding the probes, once again after 10 minutes, and one final time before leaving them for the overnight hybridization. Keep the water bath and/or the tubes covered to prevent condensation from forming in the tube during the long incubation period.
  6. Hybridize for 20-24 hours at 55° C.
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Preparation of labeled RNA probes

A key to the success of this protocol is having appropriately substituted and sized probes. With enzyme-based (alkaline phosphatase) detection, a substandard probe can still result in a strong signal because the signal development reaction can proceed for a longer time. A very good probe, on the alkaline phosphatase reaction-time scale, is one whose signal is well-developed after 10-20 minutes reaction time, and over-developed by 30 minutes; highly substituted probes with more than 1 kb of homology to a strongly expressed endogenous transcript are often over-developed in 10 minutes.

Preparing DNA Template

  1. Linearize 2-20 µg cDNA plasmid with a unique restriction site in the 5' polylinker. Only 1 µg of linearized template is required in the subsequent transcription reaction.
  2. Run out a small portion of the DNA on a gel to check for a complete restriction digest.
  3. Perform a phenol/chloroform extraction. Either
    • Extract 1x with phenol and 1x with chloroform OR
    • Extract 2x with phenol/chloroform.

    From this point on, RNase-free experimental protocols, tips, tubes, reagents, are highly recommended.

  4. Precipitate DNA with 2x volume ethanol, 0.1x volume 3 M sodium acetate, pH 5.2.
  5. Put at -20° C. for 30 minutes, then spin at maximum speed for 15 minutes in a microfuge.
  6. Wash pellet with 70% ethanol, spin at maximum speed for 2 minutes, drain completely and air dry.
  7. Resuspend in RNase-free ddH2O.
  8. For accurate quantitation, dilute a fraction of the resuspended template in TE, and take the Abs260 reading using a microcuvette in a spectrophotometer.

Synthesizing Labeled RNA

  1. Heat template DNA to 55° C. for 2 minutes, then put back on ice.
  2. Set up this reaction on ice in a RNase-free tube:
    Component Volume(µl)
    ~1 µg template DNA X
    RNase-free ddH2O Y
    10x transcription buffer 1.5
    10x hapten-U NTP mix 1.5
    RNase inhibitor 1.0
    RNA polymerase(T7, T3) 1.5
    Total 15
    Note that the reaction recipes on some product information sheets call for greater volume of reagents, but the same amount of template: 20 µl total, instead of 15 µl as shown here; many protocols call for 10 µl total.
  3. Mix thoroughly with a p20 micropipetter and incubate at 37° C. for 2 hours.
  4. Add 11 µl RNase-free ddH2O.
  5. Take 1 µl out to run on a gel, then store the reaction at -20° C. or proceed to the fragmentation step.
  6. Check the reaction product by running it on a 0.9% TAE/ethidium bromide agarose gel with a RNA marker. Although this is a typical DNA gel, the RNA product runs in a tight band at about the predicted size and should be at least 10-fold stronger than the DNA template band. See this gel for some examples of RNA probe synthesis reactions run out on such a gel.

Fragmentation and Precipitation

  1. Add 25 µl 2x carbonate buffer.
  2. Mix and incubate at 65° C. for 20-40 minutes (vary to control average probe fragment size).
  3. Add 50 µl stop solution.
  4. Add 10 µl 4 M lithium chloride.
  5. Add 5 µl 20 mg/ml tRNA (phenol/chloroform extracted, ethanol precipitated).
  6. Add 300 µl ethanol.
  7. Vortex and put at -20° C. for 30 minutes.
  8. Spin at maximum speed for 20 minutes in a 4° C. microfuge.
  9. Wash pellet with 300 µl 70% ethanol, spin at maximum speed for 2 minutes, drain completely and air dry.
  10. Dissolve pellet in 200 µl hybridization solution. The probe pellet should not be left to dry for too long, otherwise it might become hard to resuspend. Let the probe dissolve on ice for 30 minutes, then mix it thoroughly by mixing with a p200 micropipetter and vortexing. Probe stocks should be stored at -20° C.