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[…] iagen, Hilden, Germany), including a DNase treatment to digest residual genomic DNA. RNA was processed according to standard procedures and hybridized to Affymetrix GeneChip® Mouse Genome 430 2.0 microarrays. The 3 experimental replicates were hybridized independently resulting in three microarray replicates per condition. In total, 24 Affymetrix gene chips were hybridized with RNA from 72 retinas of 72 mice., To analyze the quality of the results after gene chip hybridization we employed RReporterGenerator [] combining Affymetrix-style QC, RMA and residual QC. The complete report is available in additional file ., Affymetrix raw gene expression data were summarized and normalized using the GCRMA procedure []. The data were filtered in order to remove probe-sets with constant low-level expression. Probe-sets were removed which showed replicate means for a given time-point for both treatments below the threshold separating the two peaks of the bimodal distribution of signal-intensity values. This procedure was performed independently for each of the differential testing procedures. The filtered data-sets were subsequently subjected to t-tests with multiple testing correction and control of the false discovery rate (FDR) using OCplus package [] available under Bioconductor []. By comparing the plotted number of differentially expressed genes at various FDR levels, corresponding threshold values for the maximum acceptable FDR were chosen with the aim of keeping homogenous groups with similar FDR together., To group differentially regulated genes according to their biological function the Affymetrix IDs were imported into the Database for Annotation, Visualization and Integrated Discovery (DAVID) from the National Institute of Allergy and Infectious Diseases (NIAID), NIH [,] and into Ingenuity Pathway Analyses from Ingenuity Systems []., cDNA was prepared from equal amounts of total retinal RNA, using oligo(dT) primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA). 10 ng of cDNA was amplified in a LightCycler 480 instrument (Roche Diagnostics AG, Rotkreuz, Switzerland) using LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics AG) and appropriate primer pairs [see additional file ]. mRNA levels were normalized to β-actin and relative values were calculated using a respective calibrator., Retinas were homogenized in 0.1 M Tris/HCl (pH 8.0) by sonification at […]

Pipeline specifications

Software tools GC–RMA, OCplus, DAVID