Computational protocol: Brain and Testicular Tumors in Mice with Progenitor Cells Lacking BAX and BAK

Similar protocols

Protocol publication

[…] Brains were cut mid-sagitally and distinct regions removed under a dissection microscope. Testes were flash frozen and cut in half for histology and RNA extraction. Total RNA was extracted from tissues frozen in Trizol (Invitrogen) using Qiagen RNeasy. RNA was reverse transcribed and hybridized to a mouse Gene 1.0 ST chip for the testes samples and to a mouse 430 2.0 full chip for the brain samples at the Dana-Farber Cancer Institute Microarray Core Facility. Array quality was assessed using R/Bioconductor (). All arrays passed visual inspection and no technical outliers were identified. Transcript level summarization and RMA normalization of CEL files was achieved using the Oligo package (). To identify genes correlating with the phenotypic groups, we used limma () to fit a statistical linear model to the data and then tested for differential gene expression in the contrasts of interest. Results were adjusted for multiple testing using the Benjamini and Hochberg (BH) method (). Gene set enrichment analysis (GSEA) was performed with the pre-ranked implementation of the GSEA software package() using the moderated t-statistic from limma to determine rank order. Tissue specific gene sets for the four different components of the testis were derived from the E-AFFX-5 dataset available at ArrayExpress. Briefly, each of the four testis components was first analyzed individually using Barcode() to determine the subset of genes that are expressed in that tissue, and genes that were found to be expressed in all four tissues were excluded. Barcode analysis was similarly used in deriving gene signatures for the aggressive brain tumor and brains bearing SVZ proliferations, with additional filtering criteria requiring an absolute fold-change greater than two relative to wild-type tissue. Stem cell signatures were identified from the GeneSigDB database() and were tested for significance using the CP (v2.5) gene set from MSigDB as background. […]

Pipeline specifications

Software tools Oligo package, limma, GSEA
Application Gene expression microarray analysis
Organisms Mus musculus
Diseases Brain Diseases, Neoplasms