The log2 expression level of a given promoter was calculated as the weighted average of the expression levels of all probes associated to it. Using mapping of the probes to the UCSC collection of mouse mRNAs, probes were then associated to a comprehensive collection of mouse promoters available from the SwissRegulon database (Pachkov et al., 2013). Probes were classified as expressed or non-expressed by using the “Mclust” R package and, after removal of non-expressed probes, the intensity values were quantile normalized across all samples. protein (LZIP) regulates the expression of genes involved in inflammation, cell migration, and stress response. LPS-induced renal Cox-2, loss in PGC1a and enzymatic activity of cytochrome c. Raw probe intensities were corrected for background and unspecific binding using the Bioconductor package “affy”. other, no insurance), median household income of a patients zip code. The GeneChips were scanned with an Affymetrix GeneChip Scanner 3000 7G This utility carried out compression and decompression jobs quickly in our tests while remaining light on system resources usage. GeneChips were washed and stained in the Fluidics Station 450 (Affymetrix) under FS450_0002 protocol, using the Hybridization Wash and Stain Kit (Affymetrix) The RNA was extracted using Trizol® and according to the Trizol® Reagent RNA extraction protocol.īiotinylated cDNA were prepared according to the WT Expression Kit (Ambion) followed by the WT Terminal Labeling and Hybridization Labeling Kit (Affymetrix) from 270ng total RNAģ µg of fragmented cRNA were hybridized for 17 hr at 45☌ on GeneChip Mouse Gene 1.0 ST Array. The differentiation to myotubes was induced by changing the medium to DMEM supplemented with 2% horse serum for 72h. The control transfection cells (previously transfected with the non-targeting siRNA pool) were cultivated in the presence of either GFP or PGC-1alpha adenovirus.Ĭ2C12 cells were grown to 90% confluence in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 Units/ml penicillin and 100ug/ml streptomycin. 24h after the transfection with siRNAs for Fos, Jun and Atf3 knockdown, the cells were cultivated in the presence of the PGC-1alpha expressing adenovirus for 48h. The transfection was performed using the siRNAs at a final concentration of 50nM and DharmaFECT1 transfection reagent according to the Thermo Scientific DharmaFECT Transfection Reagents siRNA Transfection Protocol. lzip is a free, command-line tool for the compression of data it employs the LempelZivMarkov chain algorithm (LZMA) with a user interface that is familiar to users of usual Unix compression tools, such as gzip and bzip2. The siRNAs for the individual knockdown of the following genes were purchased from Dharmacon (Fisher Scientific) and used to transfect myotubes: Fos, Jun, Atf3 and the non-targeting siRNA pool (control). GEO help: Mouse over screen elements for information.
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