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Genome-wide analysis of High glucose-induced gene expression by Ninjurin1 siRNA

ABSTRACT: Transcriptional profiling of Mouse cavernous primary endothelial cells (MCECs) in comparing normal glucose condition with high glucose condition or Ninjurin1 siRNA transfection condition. Objective was to determine the effect of ninjurin1 on high glucose conditioned cells on global gene expression.

ORGANISM(S): Mus musculus

PROVIDER: GSE57525 | GEO | 2014/05/20

SECONDARY ACCESSION(S): PRJNA246610

REPOSITORIES: GEO

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Genome-wide analysis of High glucose-induced gene expression by Ninjurin1 siRNA

Project description:Transcriptional profiling of Mouse cavernous primary endothelial cells (MCECs) in comparing normal glucose condition with high glucose condition or Ninjurin1 siRNA transfection condition. Objective was to determine the effect of ninjurin1 on high glucose conditioned cells on global gene expression. Total RNA obtained from MCECs, which were treated with High glucose (HG) condition or (HG+ninjurin1 siRNA). Biological replicates: 1 control replicates, 1 HG replicates, 1 (HG+ninjurin1 siRNA) replicates.

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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.

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