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Pipeline publication

[…] l inference of changes in gene expression were done using the Bioconductor package Limma in the R statistical environment. Briefly, spot and background median signals were imported into the R data-frame and normex background correction was applied. After print-tip loess within-array normalization, A-quantile normalization was used to make the signal intensities more comparable across arrays. A linear model was fitted, with coefficients estimated for all 3 possible comparisons (FSH vs Control, AREG/EREG vs Control and FSH vs AREG/EREG) and for a possible dye-effect. The empirical Bayes method to moderate the standard errors of the estimated changes in expression was applied as implemented in Limma. P-values were adjusted according to the Benjamini and Hochberg method to control for false discovery rate. Only genes with adjusted p-value <0.01 were considered significantly changed., The functional role of the differentially expressed genes was assessed for gene ontology using the freely available tool Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/)., To validate the microarray data, 50 candidate genes were selected for further analysis by real-time PCR. For this, sequence specific primers (Additional file : Table S1) were designed using Beacon Designer (Premier Biosoft, CA, USA). The total RNA was isolated from cumulus cells of 30 COCs cultured for 3 h using an RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Real-time PCR was carried out in a RotorGene 3000 cycler (Corbett Research, Sydney, Australia) using a One-Step RT-PCR Kit (Qiagen) with gene-specific primers. The 20 μl total reaction volume contained QIAGEN OneStep RT-PCR Buffer (1×), dNTP Mix (400 μM final concentration of each), reverse and forward primers (both 400 nM final concentration), SybrGreenI (0.4 μl of 1:1000 stock solution, Molecular Probes, Eugene, OR, USA), RNasine inhibitor (5 IU, Promega, Madison, WI, USA), QIAGEN […]

Pipeline specifications

Software tools limma, DAVID, Beacon Designer