Computational protocol: Identification of the principal transcriptional regulators for low-fat and high-fat meal responsive genes in small intestine

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Protocol publication

[…] Experiments were performed in duplicate by using pooled RNA from each group of mice. Total RNA (10 μg) was used for cDNA synthesis according to the Affymetrix (Santa Clara, CA, USA) manual. Hybridization to GeneChips MOE 430 v2.0 arrays representing 45,101 transcripts and expressed sequence tags followed by probing and scanning was performed according to the Affymetrix manual. The background subtraction and normalization of probe set intensities were performed using the method of Robust Multiarray Analysis described by Irizarry et al. []. To identify differentially expressed genes, gene expression intensity in HF and LF groups was compared to the fasting condition using a moderated t-test and a Bayes smoothing approach []. To correct the effect of multiple testing, the false discovery rate, was estimated from p values derived from the moderated t-test statistics []. The analysis was performed using the affylmGUI Graphical User Interface for the limma microarray package []. Genes were considered to be significantly differentially expressed if P-values were <0.05. Under these conditions, a minimal mean ratio of 2 fold was used as threshold for induced or repressed genes by the LF or HF ingestion. […]

Pipeline specifications

Software tools affylmGUI, limma
Application Gene expression microarray analysis
Organisms Mus musculus