Preparation Of Cydyelabeled Cdna Targets

1. Prepare 7-^i solution containing 1 ^g of denatured poly(A)+ RNA with 1 ng of X poly(A)+ RNA-A (TX802; Takara, Kyoto, Japan) for external control, 50 ng ^.l-1 oligo-(dT) 12-18 mer (Invitrogen, Carlsbad, CA). Incubate the annealing reaction solution for 5 min at 70°C, and then at 42°C for 1-2 min.

2. Add 8 |l of the buffer mixture containing 4 |l of

5 x Superscript first-strand buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, and 15 mM MgCl2; Invitrogen, Carlsbad, CA), 2 11l of 0.1 M DTT and 2 |l of dNTP mixture (dATP, dCTP, dGTP, each at 5 mM, and dTTP at 2 mM), 2 |l of 1 mM Cy3-dUTP or Cy5-dUTP(Amersham Pharmacia, Piscataway, NJ), 2.5 |l of 40 units |l-1 RNase inhibitor (SIN-101; Toyobo, Osaka, Japan) and 1 | l of 200 units | l-1 Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) to the annealing mixture.

3. Following incubation at 42°C for 35 min, add 100 units of Superscript II reverse transcriptase.

4. Incubate the reaction sample for an additional 35 min.

5. Following addition of 5 |l of 0.5 M EDTA, 10 |l of 1 N sodium hydroxide, and 20 |l of distilled water to stop the reaction and to degrade the template, incubate them for 1 h at 65°C.

6. Neutralize the solution with 25 |l of 1 M Tris-HCl (pH 7.5).

7. Combine the reaction products of two samples (one with Cy3 labeling and the other with Cy5 labeling).

8. Place the samples in a Microcon YM-30 microconcentrator (Millipore, Bedford, MA).

9. Add 250 |l of TE buffer, spin for 17 min in a benchtop microcentrifuge at a high speed to a volume of 10 |l, and discard the flow-through product. Repeat this step four times.

10. Collect the targets by inverting the filter and spinning for 5 min.

11. Add several microliters of distilled water to the Microcon.

12. Invert the filter, spin, and add distilled water so that the final volume of the collected targets is 18 |l.

13. Add 5.1 |il of 20 x SSC, 2.5 |il of 2 |ig |il-1 yeast tRNA and 4.8 |l of 2% SDS to the probes.

14. Denature the target samples by placing them in a 100°C heat block for 2 min, leave at room temperature for 5 min, and then use for hybridization.

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