disease state: triple negative breast cancer cell type: triple negative breast cancer cells gender: female age (y): 59
Extracted molecule
total RNA
Extraction protocol
We purified 30 TNBC, 13 normal ductal cells using lasermicrobeam microdissection system (PALM MicroBeam, Carl Zeiss MicroImaging Co., Ltd) in accordance with the manufacturer’s guidance. Dissected cancer and normal ductal cells were dissolved in RLT lysis buffer (QIAGEN, Maryland, CA) containing 1% β-mercaptoethanol. The extracted total RNAs were purified with RNeasy Mini Kit (QIAGEN, Velanica, CA) according to the manufacture’s protocol. For RNA amplification and labeling, we used Agilent Low-Input QuickAmp labeling kit according to the manufacture’s guidance. Briefly, 100 ng of total RNA of each sample was amplified T7-RNA polymerase and simultaneously incorporated Cy3-labeled CTP.
Label
Cy3
Label protocol
For RNA amplification and labeling, we used Agilent Low-Input QuickAmp labeling kit according to the manufacture’s guidance. 100 ng of total RNA of each sample and normal organs including heart, lung, liver, and kidney (Takara) was amplified T7-RNA polymerase and simultaneously incorporated Cy3-labeled CTP.
Hybridization protocol
2 μg of Cy3-labeled cRNA was fragmented and hybridized onto the Agilent Whole Human Genome Microarray 4 × 44K slide (Agilent-014850, G4110F), with rotating incubation at 65˚C for 18 h.
Scan protocol
Slides 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 immediately by brief centrifugation, and scanned by the Agilent Microarray scanner system (G2505B) in an ozone protection fume hood.
Data processing
Features of scanned image files containing the Cy3-fluorescence signals of the hybridized Agilent Microarrays were extracted using the Agilent Feature Extraction (ver. 9.5). The data files were subjected to the GeneSpring (ver. 11.5) for analysis. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
