Hybridization and Making RNA Probes

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

Hybridization of embryos with labeled RNA probes:

  1. Distribute the pre-hybridized embryos to ~50 µl per 1.5 ml eppendorf tube.
  2. Prepare the hybridization solution containing your 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 its previous performance.
  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 gentle finger tap on the tube after you've put them back in the water bath, once again after 10 minutes, and one final time before you leave for the night. Keeping the water bath and/or the tubes covered will 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:

I think one key to making a good fluorescent in situ is having a smoking hot probe. With enzyme-based (alkaline phosphatase) detection, a mediocre probe can still give you a nice stain because you just let the reaction go longer. But if you don't have very good probes, you might be disappointed with the results of this protocol. What is a very good probe on the alkaline phosphatase reaction-time scale? A probe whose signal is well-developed after 10-20 minutes reaction time, and over-developed by 30 minutes. Truly flaming hot probes are over-developed by 10 minutes. I'm following this fairly standard protocol for making probes.

Preparing and Quantitating DNA Template:

  1. Linearize 20 µg cDNA plasmid with 5' enzyme (sense strand) in 100 µl. Only 1 µg of linearized template is required in the transcription reaction, so you can digest a lot less. I start with this large digest because I'm making several probes from each template and also like to have a back-up supply.
  2. Run out 2 µl on a gel to check for complete cutting
  3. Increase volume to 200 µl with TE and add 20 µl 3 M sodium acetate, pH 5.2
  4. Perform a phenol/chloroform extraction as you like to do it. Either
    • Extract 1x with phenol and 1x with chloroform OR
    • Extract 2x with phenol/chloroform

    RNase-free begins here: tips, tubes, reagents, and take a shower before work!

  5. Precipitate DNA with 2x volume ethanol
  6. Freeze at -20° C. for >30 minutes, then spin at maximum speed for 15 minutes in a microfuge
  7. Wash pellet with 200 µl 70% ethanol, spin at maximum speed for 2 minutes, drain completely and air dry
  8. Resuspend in 30 µl RNase-free ddH2O
  9. For accurate quantitation by spectrophotometer, dilute 2 µl resuspended template in 100 µl TE, and take the Abs260 reading in a microcuvette

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 recipe on the Roche product information sheets calls 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.
  3. Mix thoroughly with p20 and incubate at 37° C. for 2-2.5 hours
  4. Add 11 µl RNase-free ddH2O
  5. Take 1 µl out to put 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 regular 0.9% TAE/ethidium bromide gel with a RNA marker. Your RNA product should run 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 good probe synthesis reactions.

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 ... optional: take out 3-5 µl to see the fragmented probe sizes on a gel
  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 freeze 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. Don't let the probe pellet dry for too long, it might become hard to resuspend. Let the probe dissolve on ice for a while, then mix it thoroughly by pipetting with a p200 and vortexing. Probe stocks should be stored at -20° C.