Transcription profiling by array of paired tumour and normal samples from colorectal cancer patients
ABSTRACT: We investigate the relevance of RNA integrity in gene expression analysis as well as analysis methods to accommodate the possible effects of degradation using paired tumour and normal samples from colorectal cancer patients undergoing colonic resection.
BACKGROUND: RNA and microarray quality assessment form an integral part of gene expression analysis and, although methods such as the RNA integrity number (RIN) algorithm reliably asses RNA integrity, the relevance of RNA integrity in gene expression analysis as well as analysis methods to accommodate the possible effects of degradation requires further investigation. We investigated the relationship between RNA integrity and array quality on the commonly used Affymetrix Gene 1.0 ST array platfo ...[more]
Project description:We investigate the relevance of RNA integrity in gene expression analysis as well as analysis methods to accommodate the possible effects of degradation using paired tumour and normal samples from colorectal cancer patients undergoing colonic resection.
Project description:We were interested in determining what genes might be controlled by TFAP2C and/or TFAP2A, either directly or indirectly through regulation of ER-alpha and potentially other signaling pathways. We performed an microarray analysis in MCF7 cells with elimination of either TFAP2C or TFAP2A. The patterns of gene expression with alteration of TFAP2 activity were compared to changes in expression induced by estrogen exposure. Knock-down of TFAP2C in the presence of estrogen altered the pattern of several known ERalpha-regulated genes and a number of genes outside the estrogen-regulated pathways. Experiment Overall Design: 6 samples were analyzed. Experiment Overall Design: 1. MCF7 cells treated with TFAP2C siRNA, without the presence of estrogen. Experiment Overall Design: 2.MCF7 cells treated with TFAP2C siRNA, with the presence of estrogen. Experiment Overall Design: 3.MCF7 cells treated with TFAP2A siRNA, without the presence of estrogen. Experiment Overall Design: 4.MCF7 cells treated with TFAP2A siRNA, with the presence of estrogen. Experiment Overall Design: 5.MCF7 cells with no siRNA treatment, without the presence of estrogen. Experiment Overall Design: 6.MCF7 cells with no siRNA treatment, with the presence of estrogen.
Project description:poly(A)+ RNA samples from hnRNP C siRNA knockdown and control HeLa cells were compared on a splice-junction microarray to detect changes in alternative splicing. Using the ASPIRE3 algorithm, we detected changes in splicing at 1,340 alternative exons. We observed a similar incidence of increased or decreased exon inclusion in hnRNP C knockdown cells, indicating that hnRNP C can either silence or enhance exon inclusion, respectively. We validated changes at 26 exons by RT-PCR with a 92% success rate.
Project description:Macrophage activation during the innate immune response is tightly regulated to prevent tissue damage while activating the defense to cellular attack. Using a mouse model where Trim33 is specifically deleted in mature myeloid cells, we show that TRIM33 is essential for two aspects of the inflammatory response in vivo. Loss of TRIM33 attenuates the initiation of macrophage activation by lipopolysaccharide (LPS) and TRIM33 is necessary to switch off transcription of inflammatory genes during late stages of LPS activation. Using chromatin immunoprecipitation coupled to deep sequencing, we provide a link between TRIM33 binding, RNA Polymerase II occupancy and H3K4me3 spreading on inflammatory genes in macrophages and reveal novel insights concerning the transcriptional regulation of Ifn-beta where TRIM33 exerts a repressive function via a distal regulatory region during late stages of LPS activation of macrophages. These findings pinpoint TRIM33 as a major regulator of the resolution of inflammation and indicate that transcriptional regulators can fine-tune H3K4me3 spreading. To study the role of TRIM33 in the transcriptional response induced by pathogen receptors, we analyzed whether lack of TRIM33 in macrophages affected the TLR-mediated regulation of proinflammatory and antimicrobial genes. To study this role, we bred TRIM33fl/fl mice with Lyz-Cre mice (obtained from The Jackson Laboratory, Bar Harbor, Maine, USA) where the Cre recombinase gene is under the regulatory sequences of the Lyz gene that is expressed only in mature myeloid cells. Bone marrow cells from 2 LyzCre/Trim33+/+ mice and 2 LyzCre/Trim33flox/flox mice were then differentiated in macrophages and treated during 0h, 4h, 12h and 24h with LPS. Total RNA was extracted from macrophages and analysed using cDNA microarrays. The set of gene expression consists of 16 samples of RNA of bone marrow derived macrophages activated with 100ng/ml of LPS during 0h, 4h, 12h, 24h, 8 samples from 2 LyzCre/Trim33+/+ mice and 8 samples from 2 LyzCre/Trim33flox/flox mice.
Project description:Profiling the genome-wide methylation status of head and neck squamous cell carcinoma and normal subjects Screening HNSCC-related candidate methylation gene by comparison of HNSCC tissues vs normal subjects.