ABSTRACT: We used state of the art mass spectrometry and RNA sequencing (RNA-Seq) to provide the first integrated proteomic, phosphoproteomic and transcriptomic atlas of Arabidopsis. For this we measured 30 different Arabidopsis tissues from adult plants, seedlings, callus and cell culture samples to an average depth of 17,603 +/- 1,317 transcripts, 14,430 +/- 911 proteins and 14,689 +/- 2,509 phosphorylation sites. In total we provide protein expression data for 66 % and transcript expression data for 90 % of the annotated protein coding genes (Araport11). For four plant organs we additionally measured consecutive growth stages (rosette leaves, 13 stages; flower, 6 stages; silique, 5 stages; seed, 5 stages).
Project description:Next-generation RNA-sequencing (RNA-seq) is rapidly emerging as the technology of choice for whole-transcriptome studies. However, RNA-seq is not a bias free technique. It requires large amounts of RNA and library preparation can introduce multiple artifacts, compounded by problems from later stages in the process. Nevertheless, RNA-seq is increasingly used in multiple studies, including the characterization of tissue-specific transcriptomes from invertebrate models of human disease. The generation of samples in this context is complex, involving the establishment of mutant strains and the delicate contamination prone process of dissecting the target tissue. Moreover, in order achieve the required amount of RNA, multiple samples need to be pooled. Such datasets pose extra challenges due to the large variability that may occur between similar pools, mostly due to the presence of cells from surrounding tissues. Therefore, in addition to standard quality control of RNA-seq data, analytical procedures for control of 'biological quality’ are critical for successful comparison of gene expression profiles. In this study, the transcriptome of the central nervous system of a Drosophila transgenic strain with neuronal-specific RNAi of a ubiquitous gene was profiled using RNA-seq. While conducting our investigation, we observed the existence of an unusual variance in the libraries and showed that the expression profile of a small panel of genes was sufficient to identify libraries with low levels of contamination from neighboring tissues, enabling the selection of a robust dataset for differential expression analysis. We additionally analyzed the potential of profiling a complex tissue to identify cell-type specific changes in response to target gene down-regulation. Finally, we demonstrated that trimming 5’ ends of reads decreases nucleotide frequency biases, increasing the coverage of protein coding genes with a potential positive impact in the incurrence of sampling errors. Central Nervous System profiles of third instar larvae WT (N=6), C24 (N=7), and X7/C24 D. melanogaster were generated by sequencing using the Illumina Hiseq2000.
Project description:Background Retinoblastoma is a pediatric eye cancer associated with RB1 loss or MYCN amplification (RB1+/+MYCNA). There are controversies concerning the existence of molecular subtypes within RB1-/- retinoblastoma. To test whether these molecular subtypes exist, we performed molecular profiling. Methods Genome-wide mRNA expression profiling was performed on 76 primary human retinoblastomas. Expression profiling was complemented by genome-wide DNA profiling and clinical, histopathological, and ex vivo drug sensitivity data. Findings RNA and DNA profiling identified major variability between retinoblastomas. While gene expression differences between RB1+/+MYCNA and RB1-/- tumors seemed more dichotomous, differences within the RB1-/- tumors were gradual. Tumors with high expression of a photoreceptor gene signature were highly differentiated, smaller in volume and diagnosed at younger age compared to tumors with low photoreceptor signature expression. Tumors with lower photoreceptor expression showed increased expression of genes involved in M-phase and mRNA and ribosome synthesis and increased frequencies of somatic copy number alterations. Interpretation Molecular, clinical and histopathological differences between RB1-/- tumors are best explained by tumor progression, reflected by a gradual loss of differentiation and photoreceptor expression signature. Since copy number alterations were more frequent in tumors with less photoreceptorness, genomic alterations might be drivers of tumor progression. Fresh frozen material from 76 primary human retinoblastoma samples were profiled with Affymetrix human genome u133 plus 2.0 PM microarray
Project description:Pyrenochaeta lycopersici causes corky root disease of tomato. A comparative RNA-Seq-based transcriptome analysis was conducted at 96 hpi (hours post infection) on two tomato cultivars: the resistant Mogeor and its genetic background, susceptible Moneymaker to investigate the differences in their transcripts and identify the molecular bases of this plant-pathogen interaction and gain hints over resistance mechanisms.
Project description:Background: DNA epigenetic modifications, such as methylation, are important regulators of tissue differentiation, contributing to processes of both development and cancer. Profiling the tissue-specific DNA methylome patterns will provide novel insights into normal and pathogenic mechanisms, as well as help in future epigenetic therapies. In this study, 17 somatic tissues from four autopsied humans were subjected to functional genome analysis using the Illumina Infinium HumanMethylation450 BeadChip, covering 486 428 CpG sites. Results: Only 2% of the CpGs analyzed are hypermethylated in all 17 tissue specimens; these permanently methylated CpG sites are located predominantly in gene-body regions. In contrast, 15% of the CpGs are hypomethylated in all specimens and are primarily located in regions proximal to transcription start sites. A vast number of tissue-specific differentially methylated regions are identified and considered likely mediators of tissue-specific gene regulatory mechanisms since the hypomethylated regions are closely related to known functions of the corresponding tissue. Finally, a clear inverse correlation is observed between promoter methylation within CpG islands and gene expression data obtained from publicly available databases. Conclusions: This genome-wide methylation profiling study identified tissue-specific differentially methylated regions in 17 human somatic tissues. Many of the genes corresponding to these differentially methylated regions contribute to tissue-specific functions. Future studies may use these data as a reference to identify markers of perturbed differentiation and disease-related pathogenic mechanisms. DNA methylation analysis of the total 72 tissue samples and controls was performed with the Illumina Infinium HumanMethylation450 BeadChip. The 17 post-mortem human somatic tissues used in this study were collected at the time of autopsy. Controls for unmethylated and methylated DNA were represented, respectively, by whole-genome amplified DNA from subcutaneous adipose tissue (using the GenomiPhi DNA amplification kit; GE Healthcare, Piscataway, NJ, USA) and the universal methylated human DNA standard (Zymo Research). We had two technical and two biological replicates processed by chip technique.
Project description:Metastasis is a complex process involving loss of adhesion, migration, invasion and proliferation of cancer cells. Cell adhesion molecules play a pivotal role in this phenomenon by regulating cell-cell and cell-matrix interactions. CD146 (MCAM) is associated with advanced tumor stage in melanoma, prostate and ovarian cancers. For this study, the MDA-MB-231 cell line was used as a prototypic mesenchymal and invasive cell line, spontaneously expressing high levels of CD146. Using whole-genome DNA microarrays, we investigated genes for which expression was modified by CD146 down-regulation, obtained by siRNA or shRNA technology Experiment Overall Design: Expression profiles of two cell lines transfected by two differents ShRNA directed against CD146 were compared with expression profiles of 3 control cell lines (i.e. native cell line, empty plasmid and shRNA directed against GFP)
Project description:Array-based DNA methylation profiling in peripheral blood leukocytes of 30 infertile men with impaired spermatogenesis as compared to 10 fertile men using the Illumina Infinium HumanMethylation 450k Bead Chip reveald 471 CpG sites (287 genes) to be differentially methylated between both groups. These CpG loci were significantly enriched for the gene ontology functions MHC class II receptor activity and piRNA binding. The latter was associated with two methylation-sensitive SNPs in the genes PIWIL1 and PIWIL2, respectivly, which showed significant allele distribution skewing in the infertile cohort. 445/471 differentially methylated CpGs were associated with SNPs, but 26 (15 genes) were not genomically templated and included the ENO1, MTA2, BRSK2 and LBX2 genes previously associated with fertility and spermatogenesis. The study identifies surrogate DNA methylation markers for idiopathic infertility in peripheral blood and suggests allele-specific DNA methylation differences at regulatory sites of genes involved in piRNA regulation to be associated with disturbed spermatogenesis. Bisulfite converted DNA of peripheral blood leukocytes from 30 infertile men and 10 fertile men as controls were hybridized to the Illumina Infinium HumanMethylation 450k Bead Chip.
Project description:In order to identify the gene targets of frequently altered chromosomal regions in retinoblastoma, a meta-analysis of genome-wide copy number alterations studies on primary retinoblastoma tissue and retinoblastoma cell lines was performed. Published studies were complemented by copy number and gene expression analysis on primary and cell line samples of retinoblastoma. This dataset includes the gene expression data of the retinoblastoma cell lines This data set contains the gene expression (Affymetrix human genome u133 plus 2.0 PM) results for 7 unique retinoblastoma cell lines. For one of the 7 unique cell lines, 3 RNA isolations were performed and were profiled on seperate arrays, adding up to 9 unique array files. Copy number data for primary retinoblastoma (tumor and blood DNA) and retinoblastoma cell lines are available (controlled-access) at the European Genomics Archive. Gene expression data of primary retinoblastoma is available under GSE59983. The GSE59983 records represent the primary tissue gene expression data and the CN data will be deposited into a controlled-access database, probably EGA.
Project description:Analysis of TF isoform-induced breast cancer cell transformation at gene expression level. The hypothesis tested in the present study was that expression of TF isoforms upregulate genes involved in proliferation and transformation, while downregulating genes involved in cell cycle arrest and apoptosis. Results provide important information on the cellular response to TF expression with respect to pro-oncogenic programs . Total RNA obtained from an MCF-7-based cell model (2A3-3) expressing full length tissue factor (2A3-3-flTF) or alternatvively spliced tissue factor (2A3-3-asTF) compared to cells expressing an empty vector control (2A3-3-pcDNA).
Project description:Autophagy is a lysosomal degradation process involved in the turnover of organelles or other cell constituents, in providing sources for energy production under starving conditions, and in cell metabolism. A key protein in the macroautophagic machinery is the autophagy related protein (Atg) 7. Constitutive deletion of Atg7 is lethal at birth. A conditional deletion of Atg7 in hepatocytes leads to hepatomegaly and in aged animals to liver tumors. With this study, we aimed at analyzing the hepatomegaly development more detailed. The 3- to 4-fold enlargement of the liver takes place between day 25 and 35 after birth (P25-P35) and persists at least until P90. This is accompanied by a change in the expression of enzymes involved in the glycogen/glucose metabolism. While glycogen synthesis is inhibited, glucose is preferentially kept as glucose-6-phosphate inside the cells, inducing a swelling of the cells caused by hyperosmolarity. An increase of lipogenic enzymes suggests that glucose-6-phosphate is delivered to lipogenic pathways, which is supported by the occurrence of a steatosis around P30. The development of hepatomegaly is accompanied by a polyploidisation of hepatocytes, an enhanced expression of genes related to inflammatory processes, and an infiltration of macrophages. Our data provide evidence that the attenuation of macroautophagy in hepatocytes leads to a glucose retention which causes cell swelling. The resulting hepatomegaly, which develops in a time interval of about 10 days, perturbs liver perfusion and induces an inflammatory reaction together with polyploidisation.