Project description:Dual-color transcriptional profiling of decoy-transfected cells, harboring AAAA[AGT]TT motif, versus scrambled-transfected cells as controls.
Project description:Transcriptional profiling of human MCF7 cells comparing mock control MCF7cells with MCF7cells transfected by siRNA against calreticulin. The latter was shown to be of having significantly decreased expression of calreticulin followed by significant decrease in migrative and invasive potentials of the MCF7 cells. Goal was to determine the effect of calreticulin knockdown on the global gene expression of MCF7 cells. Two-condition experiment, Mock control MCF7 cells vs. calreticulin siRNA knockdown MCF7 cells. Biological replicates: 3 mock control replicates, 3 transfected replicates.
Project description:Twenty million LbetaT2 cells were transfected with either control or Galphas siRNA, then were seeded in 100-mm cell culture plates in DMEM + 10% FBS. Two days later, cells were washed twice with pre-warmed PBS. Conditioned media was harvested another 24 h later, and centrifuged at 20,000 g for 10 min at 4°C to remove cell debris. To enrich secreted proteins in the conditioned media, conditioned media samples were centrifuged using Amicon centrifugal filters with a 3kDa cutoff (Millipore, Billerica, MA). A total of 8 concentrated conditioned media samples were independently prepared: 4 samples from control siRNA-treated cells, and 4 samples from Galphas siRNA-treated cells. Samples were stored at –70°C until they were sent to the Mount Sinai Proteomics Core Facility.HPLC-isobaric tags for relative and absolute quantitation mass-spectrometry (iTRAQ MS) - Data analysis ProteinPilot 3.0 (AB Sciex) was used to search the MS/MS spectra for protein identification and quantitation with its searching algorithm Paragon 3.0.0.0 (*Reference). The protein database used for searching was Uniprot mouse fasta file (release-2010_11). The search parameters include quantitation for iTRAQ 8-plex (peptide-labeled), MMTS for cysteine alkylation, trypsin for enzyme digestion, biological modifications for ID focus, and taxonomy set for Mus musculus. The detected protein threshold was set to 1.3 (95% confidence). Additionally, we converted our AB Sciex mass spectral data (TOF/TOF data) into an mzML format, using the AB Sciex MS Data Converter (beta version 1.3) tool. Finally, we used the command line tool group2xml, which is included with ProteinPilot Software, to convert the .group search engine result file to an XML file.
Project description:Transcriptional profiling of MDA-MB-231 comparing the regulatory consequences of transfecting synthetic decoys carring sRSM1 motifs versus synthetic scrambled oligos
Project description:Transcriptional profiling of MDA-MB-231 comparing the regulatory consequences of transfecting synthetic decoys carring sRSM1 motifs versus synthetic scrambled oligos Two decoy/scrambled sets, each with two biological replicates
Project description:Transcriptional profiling of human MCF7 cells comparing mock control MCF7cells with MCF7cells transfected by siRNA against calreticulin. The latter was shown to be of having significantly decreased expression of calreticulin followed by significant decrease in migrative and invasive potentials of the MCF7 cells. Goal was to determine the effect of calreticulin knockdown on the global gene expression of MCF7 cells.
Project description:ETS2 is a canonical transcriptional factor and member of the ETS family of genes. ETS2 binds to consensus ERE binding sites in a broad spectrum of genes thus affecting many intracellular molecular functions. However, the role of ETS2 in the biology and pathogenesis of lung cancers is still not known. We have found that ETS2 is down-regulated in lung tumors compared to normal lung tissue and the expression of the coding protein of the gene was a significant independent predictor of favorable outcome in NSCLC patients pinpointing to a potential tumor suppressor role for this gene. To better understand its molecular function in NSCLC, we compared and contrasted the transcriptome of lung cancer cells transfected with control siRNA and siRNA targeting ETS2. H441 lung cancer cells were transfected with SMARTpool (Dharmacon)control/scrambled siRNA or siRNA targeting ETS2. Three independent transfections were performed cells with control siRNA and for cells with siRNA specific to ETS2 where each transfection consittutes a biological replicate. Knock-down of ETS2 in all samples was confirmed by quantitative real-time PCR. Total RNA was then profiled using the Human Gene
Project description:ETS gene fusions have been characterized in a majority of prostate cancers, however key molecular alterations in ETS negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier-expression exclusively in a subset of ETS rearrangement negative cancers (~10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier-expression can be detected non-invasively in urine and observed that SPINK1 outlier-expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier-expression model, and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement negative prostate cancers. Keywords: Genetic Modification
Project description:Transcriptional profiling of human MCF7 cells comparing mock control MCF7cells with MCF7cells transfected by siRNA against calreticulin. The latter was shown to be of having significantly decreased expression of calreticulin followed by significant decrease in migrative and invasive potentials of the MCF7 cells. Goal was to determine the effect of calreticulin knockdown on the global gene expression of MCF7 cells. Two-condition experiment, Mock control MCF7 cells vs. calreticulin siRNA knockdown MCF7 cells. Biological replicates: 3 mock control replicates, 3 transfected replicates.