Total RNAs were extracted using Qiagen RNeasy Mini Kit, QIAshredder kit and RNase-Free DNase Set kit (Qiagen, Valencia, CA) following manufacturer's recommendations. The protocol includes homogenization of tissue by grinding in mortor with liquid nitrogen and binding to the RNeasy Mini spin column, then DNase were used to get rid of trace amount of DNA. RNAs were quantified using a NanoDrop-2000 spectrophotometer and their qualities were monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 100ng RNA using the One-Color Low Input Quick Amp labeling kit (Agilent, Valencia, CA) according to the manufacturer's instructions, followed by RNeasy Mini Kit (Qiagen, Valencia, CA) purification. Dye incorporation and cRNA yield were checked with the NanoDrop Spectrophotometer (NanoDrop Technologies, Inc).
Hybridization protocol
0.6 ug of Cy3-labelled cRNA (specific activity > 8 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (GPL17077) for 17 hours at 65 °C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried using Agilent stabilization and drying solution.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 1x60k array slides (Scan Area 61X21 mm, Scan resolution 3 um, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The data were analyzed by GeneSpring GX (Agilent) using one parameter of subtype, whose values are Luminal A, Luminal B, Triple Negative and normal. Genes were filtered with one-way ANOVA (p<0.05) and fold change >= 2.0.
