ABSTRACT: DNA methylation in wild type bolting plants, wild type seedlings, and ddm1 seedlings. The purpose of the McrBC methylation microarray assay is to determine which regions of a genome are methylated versus those that are unmethylated in a single Arabidopsis thanliana genotype. McrBC is a methylation-sensitive enzyme that restricts DNA only at purine-Cmethyl half sites when separated between 50bp and 3kb. A designated amount of DNA from a particular genotype is sheared to a size range of 1kb-10kb using nebulization. We restrict half of the nebulized DNA with McrBC, and the methylated fraction is then removed from the unmethylated fraction through gel purification of DNA fragments greater than 1kb.* The remaining nebulized DNA is subjected to the same gel purification scheme, but with no McrBC treatment. In a single hybridization, the untreated sample is labeled with Cy5 and the McrBC-treated sample with Cy3. Thus, after labeling and microarray hybridization, the ratio of normalized Cy5 to normalized Cy3 represents the relative methylation at the sequence represented by the spot on the microarray. Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. This SuperSeries is composed of the following subset Series: GSE1329: DNA methylation in wild-type bolting Arabidopsis thaliana plants GSE1330: DNA methylation in ddm1 seedling Arabidopsis thaliana plants GSE1331: VC133+137, DNA methylation in ddm1 seedling Arabidopsis thaliana plants GSE1332: VC134+136, DNA methylation in wild-type seedling Arabidopsis thaliana plants Refer to individual Series
Project description:Histone 3 lysine 4 and histone 3 lysine 9 methylation in wild type and ddm1 Arabidopsis thaliana seedlings. The purpose of the chromatin immunoprecipitation/microarray (ChIP/chip) experiment is to determine which regions of a genome are enriched for a particular histone modification in a single Arabidopsis thanliana genotype. Chromatin immunoprecipitation with antibodies raised against dimethyl histone-H3 lysine-9 (H3mK9) or dimethyl histone-H3 lysine-4 (H3mK4) is performed on a selected genotype. This purified DNA from each immunoprecipiation (mH3K9, mH3K4, no antibody control) is used for random amplification to increase the quantity of DNA for microarray hybridization. The amplified DNA from each experimental sample is then labeled with Cy5 and hybridized against total input DNA from the corresponding genotype, labeled in Cy3. In a single hybridization, the total input DNA serves as a baseline and is compared to the immunoprecipitated samples. Ratios of normalized signal intensities were calculated to identify enrichment of a particular sequence after immunoprecipitation, in comparison to the total input DNA. Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. This SuperSeries is composed of the following subset Series: GSE1333: EV49+50, Histone 3 Lysine 4 methylation in wild-type Arabidopsis thaliana seedlings GSE1334: Histone 3 Lysine 4 methylation in ddm1 Arabidopsis thaliana seedlings GSE1335: EV104+105, Histone 3 Lysine 4 methylation in ddm1 Arabidopsis thaliana seedlings GSE1336: Ev106+107, Histone 3 Lysine 4 methylation in WT Arabidopsis thaliana seedlings GSE1337: EV51+52, Histone 3 Lysine 9 methylation in wild-type Arabidopsis thaliana seedlings GSE1338: EV59+60, Histone 3 Lysine 9 methylation in ddm1 Arabidopsis thaliana seedlings GSE1339: Histone 3 Lysine 9 methylation in wild-type Arabidopsis thaliana seedlings GSE1340: EV110+111, Histone 3 Lysine 9 methylation in ddm1 Arabidopsis thaliana seedlings Refer to individual Series
Project description:mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings and bolting plants. Features found to be significantly enriched for DNA methylation were determined. This SuperSeries is composed of the following subset Series: GSE1324: EV23+24 mRNA levels in Wild-type versus ddm1/+ backcross bolting Arabidopsis thaliana plants GSE1325: EV33+34 mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings GSE1326: VC109+111 mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings GSE1327: EV39+40 mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana seedlings GSE1328: VC110+112 mRNA levels in Wild-type versus ddm1 bolting Arabidopsis thaliana plants Refer to individual Series
Project description:To assess changes in DNA methylation state associated with breast cancer we analyzed DNA from normal and cancerous breast specimens on CpG islands microarray using methylation-sensitive enzyme McrBC for target preparation. Cy3 was used for McrBC cut sample, Cy5 was used for reference Mock sample.
Project description:Whole-genome screening of CpG Island methylation status by array-based profiling of absolute methylation status (array-PAMS). Methylation-specifically digested DNA (Cy3) was hybridized against methylation-sensitively digested DNA (Cy5) from the same sample. CGI methylation in 20 pediatric medulloblastomas (M) and normal cerebellum (Cb, pool of five unaffected donors, age 25–33 years)
Project description:We have systematically profiled DNA methylation at promoter CpG islands (CGIs) in ovarian cancer. Epithelial ovarian tumours, excluding mucinous and clear cell cancers, prospectively collected through a cohort study, were analyzed by differential methylation hybridization (DMH) (Nouzova M et al, 2004) in duplicates. The loci targeted by the custom-designed microarray are the promoter CpG islands (Gardiner-Garden and Frommer, 1987) of the genes involved in the Wnt, p53, AKT/mTOR, BRCA1/2 and Redox pathways, DNA repair (HR, NHEJ and MMR), FA family and IgLON family. 111 ovarian tumor samples were assayed by DMH in duplicates. McrBC digested (Cy5) and undigested (Cy3) samples were competitively hybridized on the Agilent custom-designed microarrays 8x15k.
Project description:A part of current research has intensively been focused on the proliferation and metabolic processes governing biological systems. Since the advent of high throughput methodologies like microarrays, the load of genomic data has increased geometrically and along with that the need for computational methods which will interpret these data. In the present work we study in vitro the common proliferation and metabolic processes, which are combined to the common oncogenic pathways, as far as gene expression is concerned, between the T-cell acute lymphoblastic leukemia (CCRF-CEM) and the rhabdomyosarcoma (TE-671) cell lines. We present a computational approach, using cDNA microarrays, in order to identify commonalities between diverse biological systems. Our analysis predicted that JAK1, STAT1, PIAS2 and CDK4 are the driving forces in the two cell lines. This type of analysis can lead to the understanding of the common mechanisms that transform physiological cells to malignant, as well as it reveals a new holistic way to understandthe the dynamics of tumor onset as well as the mechanistics of oncogenic drivers. The present work is concerned with the common expressional profile of two cell lines: the T-cell acute lymphoblastic leukemia (CCRF-CEM) and the rhabdomyosarcoma (TE-671) cell lines. Our investigation was focused on the identification of genes that share a common expression profile between the two cell lines. Both cell lines are characterized by the fact that their differentiation has stopped at an early stage, before they mature to their final cell type. Normally, these cells would have matured and progressed into differentiated cells, constituting blood and muscle cells, respectively. At some unknown point, normal differentiation ceased for these cells and they became malignant. From that point on, to the first manifestation of symptoms of malignancy, there is a lack of knowledge regarding the mechanisms underlying oncogenesis. From these observations, the question whether two distinct cell types destined to fulfill different functions, manifest similar mechanisms of growth and progression due to their malignant character, arises. The present study was focused on the identification of the differential expression profiles underlying the two cell lines. A previous report studied the expression profile of seven ARMS cell lines possessing the PAX3-FKHR fusion gene, along with other cell lines of different tumor types (22). To our knowledge, this is the first time that a comparison between two totally different types of neoplasia, such as the CCRF-CEM and TE-671 cell lines, is attempted. These mechanisms are examined with purpose to identify common drivers that lead to the progression of tumor cells. We hereby propose a new computational approach for the investigation of common oncogenic drivers.
Project description:Elevated Met receptor tyrosine kinase (RTK) expression correlates with poor outcome in breast cancer, yet a causal role for Met in the development of breast cancer has not been directly established. To examine this question, we generated a transgenic mouse model that targets expression of an oncogenic Met receptor (MetMut) to the mammary epithelium. We show that MetMut induces mammary tumors with a variety of histopathologies that exhibit gene expression profiles sharing similarities with human basal and luminal breast tumor subtypes. Among all breast cancers, we further demonstrate that the Met receptor is primarily overexpressed in human basal and HER2 positive breast cancers, and that a Met associated gene expression signature identifies patients with poor prognosis. Experiment Overall Design: Common reference design. 26 samples (including 20 normal tissue and 32 tumor tissue samples) replicated twice as dye swaps, generating a total of 52 arrays.
Project description:Overexpression and/or amplification of the ErbB-2 oncogene, as well as inactivation of the tumor suppressor PTEN, are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the MMTV promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibit homogenous pathology. PTEN-deficient/NIC tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the PI3K/Akt signalling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal-like subtype of breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation, and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signalling pathways. Experiment Overall Design: Common reference design. 9 samples (including 2 normal tissue and 7 tumor tissue samples) replicated twice as dye swaps, generating a total of 18 arrays.