1. Equilibrate the probe array to room temperature immediately before use (probe arrays should be stored at 4°C).

2. Pre-hybridize the probe array with 1 x hybridization buffer. Keep the array upside down. Insert a clean small pipette tip into the top septum of the array to allow venting of air from the chamber inside the probe array. Pipette 200 |l of

1 x hybridization buffer into the array through the bottom septum. Incubate for at least 10 min at 45°C with 60 r.p.m. rotation (GeneChip® hybridization oven).

3. Preparing the hybridization target: mix the following components in a RNAse-free

1.5-ml Eppendorf tube:

15 |g of fragmented cRNA 37.5 |l

20 x Eukaryotic Hybridization control mix 15 |l

3 nM B2 control oligonucleotide 5 |l

Acetylated BSA (50 mg/ml) 3 |l

2 x Hybridization buffer 150 |l

RNAse-free H2O to final volume of 300 |l 86.5 |l

4. Denature the hybridization cocktail at 99°C for 5 min.

5. Incubate the hybridization cocktail at 45°C for 5 min.

6. Spin the samples at maximum speed in an Eppendorf centrifuge for 5 min to remove any insoluble material from the hybridization cocktail.

7. Remove the pre-treatment solution from the pre-hybridized probe arrays and add 200-230 |l of the hybridization cocktail into the probe array. A small air bubble inside the probe array is needed for proper mixing of the hybridization cocktail during the hybridization. Seal the septa with small 8-mm paper stickers to prevent any loss of hybridization cocktail during the incubation.

8. Hybridize for 16 h at 45°C with 60 r.p.m. rotation in the hybridization oven.

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