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Real-time quantitative PCR analysis of murine bone marrow cells

ABSTRACT: TGF-M-NM-21 signaling pathway of bone marrow of the Gata1low mouse model of myelofibrosis Four condition experiment. Biological replicates: 3 control CD1 mice, 3 Gata1low mice, 3 SB431542-treated Gata1low mice and 3 Vehicle-treated Gata1low mice.

ORGANISM(S): Mus musculus

SUBMITTER: fiorella ciaffoni

PROVIDER: E-GEOD-44797 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.

Zingariello Maria M Martelli Fabrizio F Ciaffoni Fiorella F Masiello Francesca F Ghinassi Barbara B D'Amore Emanuela E Massa Margherita M Barosi Giovanni G Sancillo Laura L Li Xiaochun X Goldberg Judith D JD Rana Rosa Alba RA Migliaccio Anna Rita AR

Blood 20130305 17

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on d ...[more]

PMID: 23462118

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Real-time quantitative PCR analysis of spleen cells

Project description:TGF-M-NM-21 signaling pathway of spleen of the Gata1low mouse model of myelofibrosis Four condition experiment. Biological replicates: 3 control CD1 mice, 3 Gata1low mice, 3 SB431542-treated Gata1low mice and 3 Vehicle-treated Gata1low mice.

2013-03-02 | E-GEOD-44795 | biostudies-arrayexpress

Real-time quantitative PCR analysis of undifferentiated rat embryonic stem cells and induced pluripotent stem cells

Project description:Validation study of a focused microarray for evaluation of undifferentiated rat ESCs.Using a focused microarray, undifferentiated embryonic stem cells (ESCs) can be distinguished from differentiated ESCs and other cells derived from the early embryo since they have a unique gene expression pattern associated with pluripotency and lack of markers of differentiation. To date, however, such an array has not been developed for the rat species and differences in genomes of rat and human or rat and mouse preclude the use of ESC focused human or mouse microarrays for the rat. Here, we developed a focused microarray for screening rat ESCs and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem cells (TS), rat extraembryonic endoderm cells (XEN), mouse embryonic fibroblast feeder cells (MEFs) and rat ESCs that have been differentiated in vitro. We used this tool to compare rat ESCs which have been expanded in a conventional rat ESC medium containing two inhibitors, e.g., GSK3 and MEK inhibitors, and leukemia inhibitory factor (LIF), and found expression of Cdx2, a gene associated with trophoblast determination. The rat ESC focused microarray described in this report has utility for rapid screening rat ESCs. This will enable optimization of culture conditions in the future. qRT-PCR gene expression profiling of undifferentiated ESCs and iPS cells. Equal amounts (1000ng) total RNA from each cell line was used in independent trials conducted by two investigators, these are technical replicates.

2014-09-25 | E-GEOD-61731 | biostudies-arrayexpress

Real-time quantitative PCR analysis of 88 microRNAs involved in human cell differentiation and development in normal fibroblasts stimulated with exogenous TGF-β1 and systemic sclerosis (SSc) dermal fibroblasts

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

Real-time quantitative PCR analysis of rat diaphragm before and after acute cervical spinal cord injury

Project description:Male Sprague Dawley adult rats were subjected to a cervical hemisection at C2 (C2HS). Costal diaphragm (both ipsilesional and contralesional sides) were assessed under control conditions (sham surgery) and at 1 and 7 days post-C2HS. We used SA Biosciences Rat Skeletal Muscle Development and Disease RT2 Profiler PCR Array to quantitate gene expression of muscle atrophy and regeneration-relevant genes from the uninjured and injured tissues on the indicated sides and at the indicated timepoints post-lesion. qPCR gene expression profiling. Tissue was derived from the costal diaphragm from control animals, and from contralesional and ipsilesional sides at days 1 and 7 post-lesion. RNA was extracted from flash-frozen tissue (TRIzol reagent) and quantitated via spectrophotometry. Equal amount total RNA from each donor was pooled prior to gene expression analysis. N=3 or 4 rats per condition. Four genes in the array study appeared to undergo unphysiologic increases in expression following SCI: Mmp9, Leptin, activin A and alpha actinin. For these genes, expression levels in control tissue were detectable but extremely low, thus fold-increases following SCI are numerically exaggerated. Therefore, we cannot explicitly comment on the physiologic scale of this response

2013-12-18 | E-GEOD-45021 | biostudies-arrayexpress

Real time quantitative PCR analysis of murine macrophages

Project description:Murine macrophages were isolated from the lungs of mice given a pulmonary challenge with C. neoformans strain H99. Mice were either given a protective (H99γ) or a mock (HKCn) immunization prior to C. neoformans H99 challenge, and macrophages were isolated from the lungs of mice 24 hours, 3 days, or 7 days post-challenge using anti-CD11b microbeads according to the Miltenyi cell sorting system. We used SA Biosciences Toll-like Receptor PCR assay panel to quantitate gene expression of signal transduction factors in total RNA isolated from macrophages derived from immunized mice compared to non-immunized. qPCR gene expression profiling. Macrophages from 5 mice per group were pooled and assayed as indicated in the summary. Each experiment was performed 3 times and the resulting Ct values of each group from each experiment averaged prior to data analysis. TIme points were analyzed separately

2012-08-09 | E-GEOD-39990 | biostudies-arrayexpress

Real-time quantitative PCR analysis of MDA MB 231 cells

Project description:MDA-MB-231 breast cancer cells were infected with either Ad-GFP, Ad-FLI1, or Ad-PDEF qPCR gene expression profiling of MDA-MB-231 breast cancer cells were infected with either Ad-GFP, Ad-FLI1, or Ad-PDEF.

2012-12-31 | E-GEOD-38499 | biostudies-arrayexpress

Real-time quantitative PCR analysis of 88 microRNAs involved in human cell differentiation and development in systemic sclerosis (SSc) dermal fibroblasts

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

The expression profiles of extracellular matrix-related genes in the presence or absence of IL-17A or -17F as measured with the PCR array in systemic sclerosis (SSc) dermal fibroblasts

Project description:We examined the effect of IL-17 signaling pathway on extracellular matrix (ECM) expression and the involvement of IL-17 signaling pathway in pathogenesis of SSc. To identify differences in the expression pattern of ECM genes in IL-17A- or IL-17F-treated cells, we performed PCR array analysis, consisting of 84 ECM-related genes. Normal human dermal fibroblasts were cultured until they were confluent, and then stimulated with IL-17A or IL-17F for 12 hours, and total RNA was extracted. A mixture of equal amounts of mRNAs from three normal fibroblasts was prepared in the presence or absence of IL-17A or -17F, and mRNA expression profile was evaluated using PCR Array. Normal fibroblasts were obtained by skin biopsies from 3 healthy donors. Fibroblasts from donors were used and treated separately as indicated in the summary. Equal amount total RNA from each donor was pooled prior to gene expression analysis.

2012-01-01 | E-GEOD-33581 | biostudies-arrayexpress

PCR array of inflammatory cytokines and receptors in mouse lungs with NF-kappaB RelA mutant in alveolar epithelial cells during LPS stimulation

Project description:A murine model of RelA mutated throughout the alveolar epithelium was generated. Mice were anesthetized and intratracheally instilled with 50M-NM-<g LPS into the left lung lobe. Mice were euthanized after 6 hours after instillation, and left lung lobes were collected to isolate RNA. We used SABioscience Mouse Inflammatory Cytokines and Receptors PCR Array to evaluate whether the expressions of lung cytokines and receptors during LPS stimulation are dependent on alveolar epithelial NF-M-NM-:B RelA or not. qPCR gene expression profiling. RNA were collected from six different mouse lungs in each genotype (wild-type and alveolar epithelial RelA mutant). Equal amount of RNA (200ng) was pooled from six different mouse lungs, and 1M-NM-<g of total RNA (1.2M-NM-<g) was used to the PCR array.

2012-08-03 | E-GEOD-38398 | biostudies-arrayexpress

PCR array of inflammatory cytokines and receptors in mouse lungs with NF-kappaB RelA mutant in alveolar epithelial cells during pneumococcal pneumonia

Project description:A murine model of RelA mutated throughout the alveolar epithelium was generated. Mice were anesthetized and intratracheally instilled with 106 CFU of Streptococcus pneumoniae serotype 3 into the left lung lobe. Mice were euthanized after 15 hours after instillation, and left lung lobes were collected to isolate RNA. We used SABioscience Mouse Inflammatory Cytokines and Receptors PCR Array to evaluate whether the expressions of lung cytokines and receptors during pneumococcal pneumonia are dependent on alveolar epithelial NF-M-NM-:B RelA or not. qPCR gene expression profiling. RNA were collected from six different mouse lungs in each genotype (wild-type and alveolar epithelial RelA mutant). Equal amount of RNA (200ng) was pooled from six different mouse lungs, and 1M-NM-<g of total RNA (1.2M-NM-<g) was used to the PCR array.

2012-08-03 | E-GEOD-38399 | biostudies-arrayexpress

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