Genome wide screening for alternative splicing events in pancreatic cancer
Ontology highlight
ABSTRACT: We used Affymetrix GeneChip® Human Exon 1.0 ST Arrays to identify alternative splicing events in 15 samples of PDAC compared to 6 non-tumor samples. Several commercial and open source software approaches for the analysis of differential splicing were tested and a subset of overlapping results was validated using RT-PCR and sequencing. Splicing variants could be validated in several genes closely related to cancer. Pathway analysis of genes predicted to be alternatively spliced revealed an enrichment of genes in categories closely related to cell-cell interactions and kinase activity.
Project description:We used Affymetrix GeneChipM-BM-. Human Exon 1.0 ST Arrays to identify alternative splicing events in 15 samples of PDAC compared to 6 non-tumor samples. Several commercial and open source software approaches for the analysis of differential splicing were tested and a subset of overlapping results was validated using RT-PCR and sequencing. Splicing variants could be validated in several genes closely related to cancer. Pathway analysis of genes predicted to be alternatively spliced revealed an enrichment of genes in categories closely related to cell-cell interactions and kinase activity. 15 samples of pancreatic ductal adenocarcinoma and 6 non tumor pancreatic samples were analyzed for alternative splicing events
Project description:Alternative mRNA splicing represents an effective mechanism of regulating gene function and is a key element to increase the coding capacity of the human genome. Today, an increasing number of reports illustrates that aberrant splicing events are common and functionally important for cancer development. However, more comprehensive analyses are warranted to get novel insights into the biology underlying malignancies like e.g. acute myeloid leukemia (AML). Here, we performed a genome-wide screening of splicing events in AML using an exon microarray platform. We analyzed complex karyotype and core binding factor (CBF) AML cases (n=64) in order to evaluate the ability to detect alternative splicing events distinguishing distinct leukemia subgroups. Testing different commercial and open source software tools to compare the respective AML subgroups, we could identify a large number of potentially alternatively spliced transcripts with a certain overlap of the different approaches. Selected candidates were further investigated by PCR and sequence analysis: out of 24 candidate genes studied, we could confirm alternative splice forms in 8 genes of potential pathogenic relevance, such as PRMT1 regulating transcription through histone methylation and participating in DNA damage response, and PTPN6, which encodes for a negative regulator of cell cycle control and apoptosis. In summary, this first large Exon microarray based study demonstrates that transcriptome splicing analysis in AML is feasible but challenging, in particular with regard to the currently available software solutions. Nevertheless, our results show that alternatively spliced candidate genes can be detected, and we provide a guide how to approach such analyses. Exon expression analysis was performed using GeneChip Human Exon 1.0 ST microarrays in 64 AML patients.
Project description:In order to identify genes with differential gene expression or alternative splicing between the groups LL-sh4, uninfected, and shGFP we study 6 hybridizations on the Human Exon 1.0 ST array using mixed model analysis of variance. 842 genes with significant gene expression differences between the groups and 1118 genes with significant exon-group interaction (a symptom of alternative splicing) were found, including 192 genes with both gene and possible splicing differences (p<0.01). Contingency table analysis of the set of studied genes and a dataset of known pathways and gene classifications revealed that the set of alternatively spliced and expressed genes were found to be significantly over-represented in groups of the GOMolFn, GOProcess, GOCellLoc, and Pathway classes (p<0.01). Algorithm ANOVA study of 6 Human Exon 1.0 ST files.
Project description:In order to identify genes with differential gene expression or alternative splicing between the groups naive and activated we study 4 hybridizations on the Human Exon 1.0 ST array using mixed model analysis of variance. 1904 genes with significant gene expression differences between the groups and 1603 genes with significant exon-group interaction (a symptom of alternative splicing) were found, including 427 genes with both gene and possible splicing differences (p<0.01). Contingency table analysis of the set of studied genes and a dataset of known pathways and gene classifications revealed that the set of alternatively spliced and expressed genes were found to be significantly over-represented in groups of the GOMolFn, GOProcess, GOCellLoc, and Pathway classes (p<0.01). Algorithm ANOVA study of 4 Human Exon 1.0 ST files.
Project description:In order to identify genes with differential gene expression or alternative splicing between the groups naive and activated we study 4 hybridizations on the Human Exon 1.0 ST array using mixed model analysis of variance. 1904 genes with significant gene expression differences between the groups and 1603 genes with significant exon-group interaction (a symptom of alternative splicing) were found, including 427 genes with both gene and possible splicing differences (p<0.01). Contingency table analysis of the set of studied genes and a dataset of known pathways and gene classifications revealed that the set of alternatively spliced and expressed genes were found to be significantly over-represented in groups of the GOMolFn, GOProcess, GOCellLoc, and Pathway classes (p<0.01). Algorithm ANOVA study of 4 Human Exon 1.0 ST files. Factorial arrangment
Project description:In order to identify genes with differential gene expression or alternative splicing between the groups LL-sh4, uninfected, and shGFP we study 6 hybridizations on the Human Exon 1.0 ST array using mixed model analysis of variance. 842 genes with significant gene expression differences between the groups and 1118 genes with significant exon-group interaction (a symptom of alternative splicing) were found, including 192 genes with both gene and possible splicing differences (p<0.01). Contingency table analysis of the set of studied genes and a dataset of known pathways and gene classifications revealed that the set of alternatively spliced and expressed genes were found to be significantly over-represented in groups of the GOMolFn, GOProcess, GOCellLoc, and Pathway classes (p<0.01). Algorithm ANOVA study of 6 Human Exon 1.0 ST files. Factorial arrangment
Project description:We studied the evolution of alternative splicing in the early stages of species divergence in the house mouse. We sequenced the testis transcriptomes of three Mus musculus subspecies and Mus spretus using Illumina technology. On the basis of a genome-wide analysis of read coverage differences among subspecies, we identified several hundred candidate alternatively spliced regions.
Project description:We compared the whole mRNA transcript expression from control and homozygous mutant Dhx32 mice by Affymetrix Mouse Exon ST 1.0 Array ST to identify alternatively spliced mRNA transcripts We include exon level expression data from the liver of three control and three Dhx32 homozygous mutant mice 6 total samples (three control and three Dhx32 homozygous mutant) were analyzed. The p values for alternatively spliced transcripts were calculated by using the algorithms of Partek Genomics Suite version 6.4, on core probes excluding probe sets with signals less than 3.
Project description:We identified PRP4 kinase-A (PRP4ka) in a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana (Arabidopsis). Prp4 kinase, which was the first spliceosome-associated kinase shown to regulate splicing in fungi and mammals, has not yet been studied in plants. Analysis of RNA-seq data from the prp4ka mutant revealed widespread perturbations in alternative splicing. A quantitative iTRAQ-based phosphoproteomics investigation of the mutant identified phosphorylation changes in several serine/arginine-rich proteins, which regulate constitutive and alternative splicing, as well as other splicing-related factors. The results demonstrate the importance of PRP4ka in alternative splicing and suggest that PRP4ka may influence alternative splicing patterns by phosphorylating a subset of splicing regulators.
Project description:We compared the whole mRNA transcript expression from control and homozygous mutant Dhx32 mice by Affymetrix Mouse Exon ST 1.0 Array ST to identify alternatively spliced mRNA transcripts We include exon level expression data from the liver of three control and three Dhx32 homozygous mutant mice