Protocols: Hybridization for cDNA Microarray

Single round RT labeling from total RNA source

Use total RNA prepared from tissue or tissue culture as template in the reaction. We typically use 100 mg total RNA for each reaction. Set up reaction as follows:

  1. In a microcentrifuge tube add 9 ml of total RNA(100 mg) and 1 ml oligodT(25) (1mg/ml) primer.
  2. Incubate at 70 C for 10 minutes and immediately put it on ice.
  3. Set up another tube and add the components as follows:
    SuperScript II 5 x reaction buffer4 ml
    0.1 M DTT2ml
    dNTP mix (2mM of dATP, dTTP and dGTP; 1mM dCTP)1ml
    Undiluted Cy3- or Cy5-dCTP1 ml
    RNase inhibitor1ml
    Mix very well and add the RNA and primer to the tube and mix well
  4. Add 1ml of SuperScript II reverse transcriptase (Gibco BRL). Mix well and quick spin it down. And incubate the reaction at 42 C for 1 hour. Add another 1 ml of SuperScript II reverse transcriptase to the tube and incubate at 42 C for one more hour.
  5. You can go to the next step or store the reaction at - 20C at this point.

Alkaline Hydrolysis of RNA Template

  1. Add 5 1ml of 500 mM EDTA.
  2. Add 10 1ml of 1 M NaOH (We store the solution for only one week).
  3. Incubate at 65 C for one hour to hydrolyze residual RNA.
  4. Cool to room temperature and add 25 ml of 1 M Tris-HCl (pH 7.5).

Probe Purification

We use MicroSpin G-50 Columns (Amersham Pharmacia 27-5330-01) and follow the manufacturer's instructions. Briefly:

  1. Vortex the column gently and spin it at 720 xg for 1 mimute.
  2. Add the reaction into the column and spin again at 720 xg for 2 minutes (using a new tube).
  3. Use speed vac. to reduce the probe solution to ~ 10 ml.


  1. For each hybridization we use: 8 mg of PolydA 40-60 (Pharmacia 27-7988-01), 10 mg of Human Cot-I DNA (GibcoBRL 15279-011) and 2 mg of yeast tRNA (GibcoBRL 15401-029).
  2. Mix the blocking reagents with the Cy-dye labeled probe.
  3. Warm up the ExpressHyb Hybridization solution (Clontech 8015-2) at 60C.
  4. Add the warmed Hyb solution to the probe mixture up to 160 ml in total and mix well.
  5. Pre-hyb the mixture at 60 C for 1 hour.
  6. Apply the solution to the array and cover with a cover slip (Fisher 125485p). We wash the cover slip by 100% ethanol before using. Hybridize at 60 C for 16 to 24 hours.


We warm up all washing solutions to 37 C before using and then proceed the washing steps at room temperature. We just dunk the slides up and down in a glass tank reservoir 20 times.

  1. First wash: 1x SSC, 0.01% SDS ( Sometimes the cover slip will not fall off easily and you may need to soak the slide in the container for a while until the cover slip fall off. When that happens, we keep the container at 37 C).
  2. Second wash: 0.2x SSC, 0.01% SDS.
  3. Third wash: 0.1x SSC (wash twice).
  4. Spin the slides in a centrifuge at 3,000 rpm for 1 minute.

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