Computational protocol: Gene expression profiling in a mouse model of infantile neuronal ceroid lipofuscinosis reveals upregulation of immediate early genes and mediators of the inflammatory response

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Protocol publication

[…] Microarray data analyses were performed using the software package BRB Array Tools [], which included Cluster 3.0, and TreeView programs. BRB Array Tools was implemented for statistical analysis of microarray data, whereas Cluster 3.0 and TreeView were used for cluster analysis, using the "average linkage" clustering algorithm (similarity of correlation-uncentered). Venn diagrams were generated using the GeneSpring GX 7.3.1 Technology Platform []. Functional enriched categories analysis was carried out using DAVID [] and Webgestalt []. Pathway analysis was performed using the MetaCore software by GeneGo [] and DAVID.Microarray data were analyzed by Significance Analysis of Microarrays (SAM) (false discovery rate, 0.05, 1000 permutations, confidence level 90%) to identify probe sets differentially expressed in PPT1 knockout as compared to wild-type mice. SAM was applied using either all 45,101 probe sets (resulting in a list of 490 significantly regulated genes) or using 5326 probe sets after filtering (resulting in 267 probe sets representing genes with a higher degree (greater than about 1.5-fold) of regulation. Pathway analysis for the more selective SAM list was performed using the MetaCore software by GeneGo []. In addition, two sample t-tests were performed between wild-type and PPT1 knockout mice at 3, 5 and 8 months of age. Probe sets having p-values of less than 0.005 were considered significant. The resulting gene lists were further used to construct a Venn diagram (in GeneSpring) [] to identify genes overlapping or non-overlapping gene sets significantly expressed at the three different ages.Unsupervised clustering of the 5326 probe sets from 18 arrays revealed 18 gender-specific genes (all previously reported as sex-linked) and further revealed that one of the nine pairs of animals (from the 8 month group) was discordant for sex. (Each experimental group consisted of one male and two females, with the exception of the 8 month knockout, which had two males and one female). This finding prompted a more detailed analysis to determine the effect of this one instance of gender discrepancy. A t-test using a "leave out one sample" analysis was performed on all combinations for 8-month old mice. In all, six t-test results were generated in this way. The t-test results (yielding lists of significantly regulated genes) were compared by Venn diagram, and were found to be indistinguishable from similar "leave one out" analyses performed using data sets from 3-month-old and 5-month-old mice, suggesting that the one instance of gender mismatch did not have a discernible impact on the overall results.An initial analysis of functional enriched categories was carried out on the filtered gene set based on the Gene Ontology (GO) database terms using DAVID []. Categories with an enrichment score yielding a p-value < 0.05 were considered significant. Analysis of the unfiltered SAM gene set (843 probe sets representing 490 genes), used in the construction of the directed acyclic graphs, was performed using Webgestalt [] to identify enriched pathways at a significance level of p < 0.001. [...] A sampling of 14 genes from the SAM list of 267 differentially-expressed genes were assayed by quantitative real-time PCR using the fluorescent dye SYBR green I. Two sets of samples were assayed-samples from the original RNA samples used for the microarray analysis (18 samples) and a validation set consisting of a further 18 samples. Total RNA (4 μg) was reverse transcribed with random hexamer primers (Invitrogen) and Superscript II (Invitrogen) according to the directions supplied by the manufacturer. The following genes were analyzed by RT-PCR: Ctsd, Serpina3n, Lgals, A2m, Lzp-s, C1qa, C4, Cap1, Fos, Gp49a, Gfap, ErdrI, Ndufs-3, and MidI. Primers sequences are shown in Additional File . The reactions were performed using the 2× Power SYBR® Green PCR Master Mix (Applied Biosystems) and 100–300 nM of primer (see Additional File for details). Assays were performed in triplicate, and analyzed using an ABI 7300 instrument (Applied Biosystems). The primers were either designed using a commercially available program (Primer Express, Applied Biosystems) or obtained from an online database (Primer Bank) []. Primer specificity was assessed by analyzing amplicon dissociation curves for each sample. The relative mRNA level was calculated using either the comparative Ct method (if the PCR amplification efficiency of the gene and β-Actin were equivalent) or the relative standard curve method (if the PCR efficiency of the gene and β-Actin were not equivalent), and normalized against one housekeeping gene (β-Actin). […]

Pipeline specifications

Software tools TreeView, GeneSpring GX, WebGestalt, MetaCore, SAM, Primer Express
Applications Gene expression microarray analysis, qPCR
Organisms Mus musculus
Diseases Neuronal Ceroid-Lipofuscinoses, Genetic Diseases, Inborn
Chemicals gamma-Aminobutyric Acid