Computational protocol: Formin1 Mediates the Induction of Dendritogenesis and Synaptogenesis by Neurogenin3 in Mouse Hippocampal Neurons

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Protocol publication

[…] Hippocampal neurons were transduced at 5 DIV using Sindbis virus bearing myc-tagged Ngn3, prepared as described before or GFP as control. After 1 h viral particles were removed and proteins were allowed to express during 16 h. Next cells were lysed and total RNA was extracted using illustra RNAspin Mini RNA isolation kit from GE Healthcare (Buckinghamshire, UK). RNA quality was analyzed using a BioAnalyzer (Agilent Technologies, Santa Clara, CA). Differential gene expression analysis between the experimental populations (neurons expressing 6xmyc-Ngn3) and the corresponding control populations (neurons expressing GFP) was performed using one-color CodeLink Whole Mouse Genome Bioarrays (GE/Amersham, Piscataway, NJ, now Applied Microarrays, Tempe, AZ) according to manufacturer's recommendations. In order to obtain results with statistical significance, four biological replicates were analyzed per experimental group; therefore 8 RNA samples were analyzed. Hybridized arrays were scanned on an Agilent Microarray Scanner (G2565BA, Agilent Technologies) and CodeLink Expression Analysis software was used for primary data extraction from bioarray images. All data from the DNA microarray analyses are MIAME compliant and have been deposited in the Gene Expression Omnibus (GEO) data repository under the GEO accession number [GEO: GSE26911]. Microarray data were analyzed using the R language and packages from the Bioconductor project (http://www.bioconductor.org/). The codelink package was used for preprocessing the arrays, genefilter for data filtering and limma for statistical analysis. For preprocessing, background was corrected using the normexp method and quantile normalization was performed. Data were considered in the log2 scale. P-values were adjusted to control the False Discovery Rate (FDR) using the Benjamini and Hochberg correction . Genes with a Benjamini-Hochberg adjusted p-value smaller than 0.05 were selected as differentially expressed.Functional analysis was performed using Ingenuity Pathway Analysis (Ingenuity Systems┬«, www.ingenuity.com). This analysis identified the functions and/or diseases that were most significant to the dataset. Genes from the dataset that were associated with biological functions and/or diseases in the Ingenuity knowledge base were considered for the analysis. Fischer's exact test was used to calculate a p-value determining the probability that each biological function and/or disease assigned to the data set is due to chance alone. […]

Pipeline specifications

Software tools Codelink, genefilter, limma, IPA
Databases GEO
Application Gene expression microarray analysis
Organisms Mus musculus