Myeloid Leukemia

Classification of AML by DNA-Oligonucleotide Microarrays 223
Table 6
Components for the Hybridization Cocktail
Component Volume Final concentration
Fragmented cRNA 15 µg 0.05 µg/µL
Control oligonucleotide B2 (3 nM) 5 µL 50 pM
Eukaryotic hybridization controls, 20X 15 µL 1.5, 5, 25, 100 pM,
respectively
Herring sperm DNA (10 mg/mL) 3 µL 0.1 mg/mL
Acetylated bovien serum albumin (50 mg/mL) 3 µL 0.5 mg/mL
Hybridization buffer, 2X concentrated 150 µL 1X
Water Add to 300 µL
Final volume 300 µL
a microcentrifuge to pellet any insoluble material from the hybridization mixture.
4. Meanwhile, wet the microarray by filling it through one of the septa with 200 µL
1X hybridization buffer using a micropipettor and appropriate tips (Rainin).
Incubate the filled microarray in the hybridization oven for 15 min at 45°C with
constant rotation (60 rpm).
5. After 15 min, remove the buffer solution from the microarray cartridge and fill
with 200 µL of the clarified hybridization cocktail, avoiding any pelleted, insoluble
matter at the bottom of the tube.
6. Place the microarray into the hybridization oven and incubate for 16 h at 45°C
with constant rotation (60 rpm).
3.3.8. Microarrays
The U133 set (HG-U133A and HG-U133B) is a widely used design of gene
expression microarrays. A detailed description regarding sequences and probe
selection rules is available as a technical note from the manufacturer
(www.affymetrix.com).
AFFYMETRIX HG-U133A AND HG-U133B MICROARRAYS
The U133 two-array set provides comprehensive coverage of well-substantiated
genes in the human genome. It can be used to analyze the expression
level of 39,000 transcripts and variants, including greater than 33,000 human
genes. The two arrays comprise more than 45,000 probe sets and 1 million
distinct oligonucleotide features. The sequences from which these probe sets
were derived were selected from GenBank, dbEST, and RefSeq. The sequence
clusters were created from the UniGene database (Build 133, April 20, 2001)
and then refined by analysis and comparison with a number of other publicly