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:
- In a microcentrifuge tube add 9 ml of total RNA(100 mg) and 1 ml
oligodT(25) (1mg/ml) primer.
- Incubate at 70 C for 10 minutes and immediately put it on ice.
- Set up another tube and add the components as follows:
Mix very well and add the RNA and primer to the tube and mix well
|SuperScript II 5 x reaction buffer||4 ml|
|0.1 M DTT||2ml|
|dNTP mix (2mM of dATP, dTTP and dGTP; 1mM dCTP)||1ml|
|Undiluted Cy3- or Cy5-dCTP||1 ml|
- 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.
- You can go to the next step or store the reaction at - 20C at
Alkaline Hydrolysis of RNA Template
- Add 5 1ml of 500 mM EDTA.
- Add 10 1ml of 1 M NaOH (We store the solution for only one week).
- Incubate at 65 C for one hour to hydrolyze residual RNA.
- Cool to room temperature and add 25 ml of 1 M Tris-HCl (pH 7.5).
We use MicroSpin G-50 Columns (Amersham Pharmacia 27-5330-01) and
follow the manufacturer's instructions. Briefly:
- Vortex the column gently and spin it at 720 xg for 1 mimute.
- Add the reaction into the column and spin again at 720 xg for 2
minutes (using a new tube).
- Use speed vac. to reduce the probe solution to ~ 10 ml.
- 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).
- Mix the blocking reagents with the Cy-dye labeled probe.
- Warm up the ExpressHyb Hybridization solution (Clontech 8015-2) at 60C.
- Add the warmed Hyb solution to the probe mixture up to 160 ml in
total and mix well.
- Pre-hyb the mixture at 60 C for 1 hour.
- 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.
- 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).
- Second wash: 0.2x SSC, 0.01% SDS.
- Third wash: 0.1x SSC (wash twice).
- Spin the slides in a centrifuge at 3,000 rpm for 1 minute.
The University of Texas MD Anderson Cancer Center
1515 Holcombe Blvd, Houston, TX 77030
1-800-392-1611 (USA) / 1-713-792-6161