DNA damage Signaling Pathway PCR Array to profile the expression of human genes regulating in the DNA damage response (DDR) network
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Steven Grant
PROVIDER: E-GEOD-77560 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Human DNA Damage Signaling Pathway RT2 Profiler Array Kit (Qiagen, Valencia, CA) was used to profile the expression of key genes involved in the DDR network as per the manufacturer’s instructions.
2016-02-05 | GSE77560 | GEO
Project description:Full title: Altered levels of MOF (member of MYST family histone acetyl transferase) and decreased levels of H4K16ac correlate with a defective DNA damage response (DDR). The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that altered levels of H4K16ac correlate with a defective DNA damage response (DDR) to ionizing radiation (IR). The defect however is not due to altered expression of proteins involved in DDR. Specific inhibition of H4K16ac deacetylation in MOF-depleted cells rescued IR-induced abrogation of DDR. MOF was found associated with DNA-dependent protein kinase catalytic subunit (DNAPKcs), a protein involved in non-homologous end joining (NHEJ) repair, whose ATMdependent IR-induced phosphorylation was abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased the repair of DNA double-strand breaks (DSBs) by NHEJ and homologous recombination (HR). In addition, the MOF protein activity associates with chromatin upon DNA damage and colocalizes with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac, plays a critical role in the cellular DNA damage response. Keywords: Cell type comparison HEK293 cells were transfected with plasmids encoding hMOF for over-expression of the histone acetyl transferase that leads to elevated levels of acetylation of Lysine 16 of histone H4. siRNA mediated knock-down of hMOF was performed to deplete the H4K16ac levels. Total RNA samples for expression profiling was obtained from wild type (293 cells without any treatment), hMOF over-expressed and hMOF knock-down 293 cell lines. Each sample was analyzed in triplicates using EGPF dsRNA treated samples as control.
2010-03-23 | E-GEOD-20193 | biostudies-arrayexpress
Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Normal and scleroderma fibroblasts were serum-starved for 24 hours and incubated in the presence or absence of TGF-β1 (2ng/ml) for 6 hours. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
2012-04-01 | E-GEOD-34827 | biostudies-arrayexpress
Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
2012-01-01 | E-GEOD-34142 | biostudies-arrayexpress
Project description:The transcriptional profile of the human multiple myeloma (MM) cell line MM.1S treated with MLN4924 vs control MM.1S cells was characterized by oligonucleotide microarray analysis, using the human U133 plus 2.0 Affymetrix GeneChip, according to previously described protocols for total RNA extraction and purification; cDNA synthesis; in vitro transcription reaction for production of biotin-labeled cRNA; hybridization of cRNA with U133plus2.0 Affymetrix gene chips; and scanning of image output files. Scanned image output files were analyzed using DNA-Chip Analyzer (dChip) (www.dchip.org), including conversion to DCP files, normalization and modeling. The gene expression profile of MM.1S cells for each time point of MLN4924 treatment was compared to the profile of control MM.1S cells. The NEDD8 activating enzyme (NAE) is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the first step in the neddylation pathway. NEDD8 modification of cullins is required for ubiquitination of cullin-ring ligases (CRLs), which regulate degradation of a distinct subset of proteins. The more targeted impact of NAE on protein degradation prompted us to study MLN4924, an investigational NAE inhibitor, in preclinical multiple myeloma (MM) models. In vitro treatment with MLN4924 led to dose-dependent decrease of viability in a panel of human MM cell lines. In this analysis, we evaluated the molecular changes triggered in MM.1S myeloma cells by their in vitro treatment with MLN4924. The transcriptional profiles of each experimental condition were characterized by oligonucleotide microarray analysis, using the human U133 plus 2.0 Affymetrix GeneChip. The human multiple myeloma (MM) cells MM.1S were cultured in vitro in the presence or absence of the NEDD8 activating enzyme (NAE) inhibitor MLN4924 for 8, 16 or 24hrs. The gene expression profiles of drug treated cells were compared with the profiles of control MM.1S cells.
2012-05-01 | E-GEOD-33577 | biostudies-arrayexpress
Project description:Full title: Altered levels of MOF (member of MYST family histone acetyl transferase) and decreased levels of H4K16ac correlate with a defective DNA damage response (DDR). The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that altered levels of H4K16ac correlate with a defective DNA damage response (DDR) to ionizing radiation (IR). The defect however is not due to altered expression of proteins involved in DDR. Specific inhibition of H4K16ac deacetylation in MOF-depleted cells rescued IR-induced abrogation of DDR. MOF was found associated with DNA-dependent protein kinase catalytic subunit (DNAPKcs), a protein involved in non-homologous end joining (NHEJ) repair, whose ATMdependent IR-induced phosphorylation was abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased the repair of DNA double-strand breaks (DSBs) by NHEJ and homologous recombination (HR). In addition, the MOF protein activity associates with chromatin upon DNA damage and colocalizes with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac, plays a critical role in the cellular DNA damage response. Keywords: Cell type comparison
2010-03-15 | GSE20193 | GEO
Project description:Our previous findings suggest that the nucleus of the solitary tract (NTS), a pivotal region for regulating the set-point of arterial pressure, exhibits abnormal inflammation in pre-hypertensive and spontaneously hypertensive rats (SHRs) together with elevated anti-apoptotic and low apoptotic factor levels compared with that of normotensive Wistar–Kyoto (WKY) rats. Whether this chronic condition affects neuronal growth and plasticity in the NTS remains unknown. To unveil the characteristics of the neurodevelopmental environment in the NTS of hypertensive rats, we investigated the gene expression profile of neurotrophins and their receptors in SHRs compared to that of normotensive rat WKY. The NTS was dissected from the brain of 6 SHRs and 6 WKY rats and the total RNA was extracted. In both groups of rats (SHRs & WKY rats, n = 6 each), a total of 2 ug mRNA extracts from each NTS were pooled together, treated with RNase-free DNAse I (Invitrogen Life technologies) to remove any genomic contamination, and further purified using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was subsequently performed on 1 ug total RNA using SuperArray’s RT2 First Strand Kit (SABiosciences); the resulting cDNA was submitted for real-time quantitative PCR reactions on RT2 ProfilerTM PCR array plates using Superarray RT2 SYBR Green qPCR Master Mix (SAbiosciences) and iCycler iQ thermal cycler (Bio-rad), following the manufacturer’s instructions. The experiment was performed in duplicate in each group.
2015-04-15 | E-GEOD-67874 | biostudies-arrayexpress
Project description:To identify dysregulated miRNAs in PAH HPASMC, we compared the miRNA expression profiles between normal and PAH HPASMC using the RT2 miRNA PCR Array System. Total RNA was isolated using a miRNeasy Mini Kit (Qiagen, Valencia, CA) and treated with an RNase-Free DNase Set (Qiagen). After quantification with Nanodrop 2000 spectrophotometer (ThermoScientific, Rockford, IL), miRNAs were reversely transcribed using a RT2 miRNA First Strand Kit (SABiosciences, Frederick, MD). The RT2 miRNA PCR Array System (SABiosciences) was used to study miRNA profiling in normal and PAH HPASMC. U6, SNORD44, SNORD47 and SNORD48 were used as internal controls for the profiling. HPASMC isolated from oe normal donor (C128, as control), one IPAH patient (B157), one PAH patient with Eisenmenger’s syndrome (CC-008) and one PAH patient with Scleroderma (CCF-004) were used for miRNA PCR array study. Data is normalized to the control sample. Three independent sample preparations were used for this study.
2014-02-28 | E-GEOD-55427 | biostudies-arrayexpress
Project description:Real-time quantitative PCR analysis of human epithelial cells The activity of Malva sylvestris extract and fractions infected by A. actinomycetemcomitans were investigated using an adapted dual chamber model, oral human epithelial cells were used in the co-culture model. One microgram of RNA was converted in cDNA using RT2 First Strand Kit. 84 genes were analyzed using inflammatory response & Autoimmunity Array RT2 profiler (Qiagen Sabiosciences, Valencia, CA, USA) with buffers supplied by the manufacturer qPCR gene expression profiling. Oral human epithelial cells were infected by A. Actinomycetemcomintans and treated with Malva sylvestris extract and fractions prior to gene expression analysis.
2015-08-27 | E-GEOD-72443 | biostudies-arrayexpress
Project description:CLL cells obtained from patients with CLL were treated with MLN4924 to determine whether NFkB transcription targets were affected by the drug. CLL cells were treated with 1 microM MLN4924 or vehicle control for 24 hours. Cells were collected by centrifugation and B-cell were purified using negative selection (B cell purification Kit, Multenyi Biotech). RNA was isolated and submitted for Gene expression analysis
2013-03-05 | E-GEOD-44864 | biostudies-arrayexpress