disease-state: normal (reduction mammoplasty) tissue-type: breast epithlieum patient-id: 350 age-at-biopsy-years: 47
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from laser-capture microdissected tissue using the Picopure RNA isolation kit from Arcturus.
Label
biotin
Label protocol
A dual-round amplification procedure was performed on 100 nanograms total RNAusing the MessageAMP aRNA kit from Ambion. In the second round, biotin-labeled cRNA was generated from the double-stranded cDNA template using a nucleotide mix that contained biotinylated CTP and UTP (Enzo RNA Transcript Labeling Kit; Enzo Diagnostics, Farmingdale, NY). The biotinylated cRNA was purified using RNeasy affinity columns (Qiagen, Valencia, MD).
Hybridization protocol
For each GeneChip, 20 micrograms of the labeled product was fragmented in 40 mM Tris-acetate, pH 8.1, 100mM KOAc, 30mM MgOAc, for 35 minutes at 94 degrees-Celsius, to an average size of 35 to 200 bases. 15 micrograms of this fragmented, biotinylated cRNA, along with hybridization controls supplied by the manufacturer (Affymetrix), were hybridized to the arrays for 16 hours at 45 degrees-Celsius and 60 rpm. Arrays were washed and stained according to the standard Antibody Amplification for Eukaryotic Targets protocol (Affymetrix).
Scan protocol
The stained GeneChip arrays were scanned at 488 nm using an Affymetrix Gene Chip Scanner 3000 (Affymetrix, Santa Clara, CA).
Description
reduction mammoplasty histologically normal breast epithlieum patient 350
