P-CAGE-26291 - P-CAGE-26291
This protocol was performed by the Genomics Facility at Centro Nacional de Biotecnologia, CSIC (Madrid, SPAIN).
Background correction and normalization of expression data were performed using LIMMA.LIMMA is part of Bioconductor, an R language project. First, the data set was filtered based on the spot quality. A strategy of adaptive background correction was used that avoids exaggerated variability of log-ratios for low-intensity spots. For local background correction the "normexp" method in LIMMA to adjust the local median background was used. The resulting log-ratios were print-tip loess normalized for each array. To have similar distribution across arrays and to achieve consistency among arrays, log-ratio values were scaled using as scale estimator the median-absolute-value.
Linear model methods were used for determining differentially expressed genes. Each probe was tested for changes in expression over replicates by using an empirical Bayes moderated t statistic. To control the false discovery rate p-values were corrected by using the method of Benjamani and Hochberg. The expected false discovery rate was controlled to be less than 5%.