Project description:Efficient and sustained knockdown of DNMT1 transcript and protein was achieved using a consecutive transfection protocol in the mouse pituitary adenoma cell line, AtT-20. Genome wide microarray analysis identified 91 transcripts that were significantly differentially expressed relative to cells treated with a non-targeting control. Keywords: Differential expression AtT-20 cells were repeatedly treated with 20 nM siRNA (siDNMT1 or siNT) over an eight day period. After eight days total RNA was extracted and prepared for microarray analysis. The procedure was repeated three times independently.
Project description:Efficient and sustained knockdown of DNMT1 transcript and protein was achieved using a consecutive transfection protocol in the mouse pituitary adenoma cell line, AtT-20. Genome wide microarray analysis identified 91 transcripts that were significantly differentially expressed relative to cells treated with a non-targeting control. Keywords: Differential expression
Project description:Genome wide expression profiling to determine the overlap of Affymetrix-signals with SOLID sequencing RNA was extracted using the Qiagen RNeasy kit following the manufacturers guidelines, arrays were prepared and hybridized following the Affymetrix protocol.
Project description:30 µg proteins per sample were reduced (20 mM DTT Sigma, room temperature RT, 1 h) and S-alkylated (50 mM IAA Sigma, 1 h, dark). The remaining IAA was quenched with 20 mM DTT for 1 h in the dark. The proteins were digested with 1:50(w/w enzyme:protein) MS-grade trypsin/lys-C mix (Thermo Scientific) for 24 h at RT. The enzyme digestion was quenched by lowering the pH with formic acid (Fisher) and the sample was desalted with ZipTip C18 (Merck-Millipore) column. Samples were resuspended in 10 µl of 0.2% formic acid prior LC-MS/MS analysis.
