Computational protocol: COOPERATIVITY OF THE MUC1 ONCOPROTEIN AND STAT1 PATHWAY IN POOR PROGNOSIS HUMAN BREAST CANCER

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Protocol publication

[…] RNA purification and hybridization with GeneChip® Rat Genome 230 2.0 Arrays (Affymetrix, Santa Clara, CA) was performed as described (). The selection and analysis of genes differentially expressed in 3Y1/vector and 3Y1/MUC1-CD cells in vitro was based on previously detailed approaches (; ). 3Y1/MUC1-CD cells were grown subcutaneously in female athymic mice and the tumors were harvested for RNA extraction for analysis of gene expression as described (; ). Briefly, each array was hybridized with a pooled sample normalized to total RNA and consisting of RNA obtained from 3 independent xenografts or cell lines. Data were normalized using “global median normalization” across the entire dataset () and filtrated using multi-step filtration (). Subsequent analysis was based on pair-wise comparisons (3Y1/vector in vitro vs. 3Y1/MUC1-CD in vitro and 3Y1/MUC1-CD in vitro vs. 3Y1/MUC1-CD in vivo) of duplicated arrays using Significance Analysis of Microarrays (SAM) (). Differentially-expressed probe set IDs were selected using a 2.0-fold induction cut-off level with a False Discovery Ratio set to 0. Selected probe set IDs were annotated and functionally designated using Ingenuity Pathways Analysis (IPA, Ingenuity Systems, Inc., Redwood City, CA, USA). Fisher’s exact test was used to estimate the significance of functional networks within a given experimental dataset. This method estimates the likelihood that Network Eligible Molecules from an experimental dataset are linked in a specific network by random chance. A P-value ≤ 0.05 is considered significant and indicates a non-random enrichment of an experimental dataset by members of a specific functional network. […]

Pipeline specifications

Software tools SAM, IPA
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Breast Neoplasms