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Samples used for molecular studies were selected, carefully avoiding necrotic areas, fat and normal tissue and immediately snap-frozen in liquid nitrogen and stored at ?80��C until used. All tumor samples were evaluated by a pathologist and an adjacent section was stained and used for defining the percentage of tumor cells. Only specimens with more than 70% of tumor cells were included in the study. Total RNA was extracted from 100 mg of tissue using Trizol (Life Technologies, Frederick, MD, USA) following the manufacturer's instructions. Sirolimus Integrity of the RNA was checked on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). miRNA profiling was performed Biperiden HCl in the Functional Genomics core facility at INT using the miRNA expression profiling kit (Illumina, S. Diego, CA). This assay is an adaptation of the proven DASL (cDNA-mediated Annealing, Selection, Extension, and Ligation) assay. 800 ng of total RNA were retrotranscribed, annealed with a miRNA-specific oligonucleotide pool consistsing of a universal PCR priming site at the 5�� end, an address sequence complementary to a capture sequence on the BeadArray and a microRNA-specific sequence at the 3�� end. After PCR amplification and fluorescent labeling, the probes were hybridized on miRNA expression profiling_v2 BeadChips, containing 1,146 sequences for detecting 95% of miRNAs described in the miRBase database (v14.0). Raw data were normalized using the Robust Spline Normalization algorithm implemented in the lumi R package. Probes with a detection p value RAD001 mouse differences among the various studies (e.g., patient cohort, microarray platform, sample processing protocol, etc.). We downloaded breast cancer gene expression datasets with clinical information from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo). Figure options Download full-size image Download high-quality image (180 K) Download as PowerPoint slideThe table reports the complete list of the datasets used in this study and their sources. In all studies, raw data (e.g., CEL files) were available for all samples and detailed clinical information could be acquired for any analyzed sample.