Excised tumor specimens were formalin fixed paraffin embedeed in a routine process.
Extracted molecule
total RNA
Extraction protocol
Tumor areas were identified by ER immunostaining with a hematoxylin counter stain. Total RNA was isolated from 3 to 5 five μn unstained sections using the Ambion RecoverAll™ Total Nucleic Acid Isolation Kit (Applied Biosystems, Foster City, CA) and quantified using UV absorption on a spectrophotometer (NanoDrop® ND-1000). 100-200 nanograms of total RNA were converted to cDNA and amplified using a commercial service provided by Rubicon Genomics, Inc. (Ann Arbor, Mi), using the TransPlex™ Whole Transcriptome Amplification (WTA) kit.
Label
Cy3
Label protocol
3 μg of the WTA product were labeled with a fluorescent dye (Cy3) using a direct chemical labeling method (ULS™ Labeling Kit with Cy3 for Agilent Gene Expression Arrays, Kreatech Biotechnology, Amsterdam, The Netherlands)
Hybridization protocol
After the labeling procedure, the amplified cDNA was purified with the KREA pure columns (Kreatech Biotechnology, Amsterdam, The Netherlands) to remove the dye in excess and was then fragmented using the Affymetrix fragmentation buffer provided with the GeneChip® Sample Cleanup Module Kit (Affymetrix, Santa Clara, CA). Finally, the cDNA was purified with the Clean-up columns, provided as well with the GeneChip® Sample Cleanup Module Kit, an added to the Hybridization Cocktail. The cocktail was then subjected to an overnight pre-hybridization to an used Affymetrix Genechip U133 2.0 Plus. The day after, 900 ng of the unbound labeled cDNA mix removed from the chip were processed as raccomanded by the manufacturer and hybridized to the Agilent Whole Human Genome (4 X 44 k) array. The hybridization was performed at 65 °C for 17 hours at a rotation of 10 revolutions per minute.
Scan protocol
The slides were scanned using a DNA Microarray Scanner ( Agilent Technologies, Santa Clara, CA) and features were extracted using Agilent’s Feature Extraction Software (v 9.5.3.1).
Description
n/a
Data processing
Single channel hybridization intensity data were compiled using commercial software (Partek Genomic Suite, St Louis, Missouri). After quantile normalization, genes with mean intensity below 500 were filtred out. Data were log2 transformed for statistical analysis.
