Project description:Vascular Endothelial Growth Factor (VEGF) is a critical regulator of pulmonary Th2 inflammation but the underlying mechanism and the roles of miRNAs in this process have not been defined. We analyzed the effect of VEGF on lung microRNAs by microarray analysis and validated the findings by Taqman qRT-PCR. We compared the levels of microRNAs in the lungs of transgenic mice with their wild type litters after overexpression of VEGF from a lung epithelium-restricted transgene. VEGF was overexpressed in the lung epithelium of CC10-rtTA-VEGF transgenic mice by adding Doxycycline to their drinking water for 7-10 days. RNA from the lungs of VEGF transgenic mice and their wild type litters were extracted and analyzed by microarray. The expression levels of microRNAs that were significanly altered were validated in another group of mice. Lung RNA from each transgenic mouse was labeled with Cy5 and lung RNA from wild type mice were labeled with Cy3. The two labeled RNAs were hybridized to the miRNA array. This array (chip ID 01_M9.1_070362) detects miRNA transcripts listed in Sanger miRBase Release 9.1 (http://www.sanger.ac.uk/Software/Rfam/mirna/). Multiple control probes are included in each chip. The control probes are used for quality controls of chip production, sample labeling and assay conditions. Among the control probes, PUC2PM-20B and PUC2MM-20B are the perfect match and single-based match detection probes, respectively, of a 20-mer RNA positive control sequence that is spiked into the RNA samples before labeling. One may assess assay stringency from the intensity ratio of PUC2PM-20B and PUC2MM-20B, which is normally larger than 30. When the option for custom probes is selected, custom probes are also included. After hybridization three imaged were obtained. From Cy3 and Cy5 images one may directly read miRNA profiles and from Ratio images one may get a quick sense of differential expressions between the corresponding samples. The images are displayed in pseudo colors so as to expand visual dynamic range. In the Cy3 and Cy5 intensity images, as signal intensity increases from 1 to 65,535 the corresponding color changes from blue to green, to yellow, and to red. In the Cy3/Cy5 ratio image, when Cy3 level is higher than Cy5 level the color is green; when Cy3 level is equal to Cy5 level the color is yellow; and when Cy5 level is higher than Cy3 level the color is red.
Project description:The salamander microRNA expression between mid-bud limb regenerating blastemas (17 days post amputation) and non-regenerating stump tissues was compared by microarray analysis. LC Sciences arrays: Six paired samples were analyzed: three mid-bud 17dpa blastemas (bl), and three non-regenerating stumps (st). Three arrays were hybridized comparing two paired samples each time. Biological dye-swaps were made by labeling bl samples once with Cy3 and twice with Cy5; st samples were labeled accordingly twice with Cy5 and once with Cy3. Multiple arrays averaged into a single Sample record. Supplementary files: GSE29727_LC_MultiArray_SimpleNormalizedData.txt.gz GSE29727_LC_signal_ratio_mean_SD.txt.gz GSE29727_LC_t-Test_st-vs-bl.txt.gz Exiqon arrays: Six paired samples were analyzed: three mid-bud 17dpa blastemas (bl), and three non-regenerating stumps (st). Six arrays were hybridized comparing each sample labeled with Hy3 against a common reference sample made by pooling all the samples and labeling it with Hy5. Multiple arrays not averaged, represented as multiple Sample records. Supplementary file: GSE29727_Exiqon_matrix_complete.txt
Project description:Transcriptional response of Bacillus subtilis to ramoplanin in wild-type CU1065. Bacillus subtilis CU1065, WT (-RAM) vs. (+RAM) and liaR (yvqC) deletion (-RAM) vs. (+RAM). The experiment was conducted in triplicate using three independent total RNA preparations. Combined reference RNA was labeled with Cy3 and ramoplanin treated/untreated samples were labeled with Cy5.
Project description:Heme Deficient or Wild Type (normal heme) for 6 hrs with iron chelation by BPS. Single Experiment with Probe Reversal 1. GSM78518 (Cy3) vs GSM78519 (Cy5) 2. GSM78520 (Cy3) vs GSM78521 (Cy5)
Project description:The study was carried out to identify the genes regulated upon IGFBP2 modulation in breast cancer. This will help to understand the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. C5 (Cy5) Vs. Vector(Cy3), C5 (Cy5) Vs. Vector(Cy3), C12 (Cy5) Vs. Vector(Cy3), C12 (Cy5) vs. Vector(Cy3)
Project description:Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclinical tests fail in these patients, emphasizing the need of appropriate preclinical models to test novel anticancer strategies. We developed four orthotopic mouse models employing primary human PDAC cells expressing Firefly and Gaussia luciferases, enabling bioluminescence monitoring of tumor growth and metastasis formation. Additional tumor characterization was performed using MR and high frequency ultrasound imaging. Genomic and immunohistochemical analysis revealed c-Met amplification and overexpression in one of four models. Analysis of c-Met inhibitors in vitro showed that crizotinib had the most potent effect. Moreover, we demonstrated synergistic effects between crizotinib and gemcitabine – the standard of care therapeutic in PDAC patients - in vitro and in vivo. Importantly, crizotinib reduced the cytidine deaminase activity in PDAC cells causing prolonged activity of gemcitabine due to diminished metabolic inactivation, as measured by LC-MS/MS. This might at least in part explain the observed prolonged survival of concomitantly treated mice with PDAC tumors and metastases. In conclusion, our orthotopic PDAC models enabled PDAC tumor imaging, and showed genetic, histopathological and metastatic features similar to their originator tumors. This allowed the identification of c-Met as a potential therapeutic target in PDAC, and revealed a cytidine deaminase-mediated synergistic mechanism between crizotinib and gemcitabine, a combination of drugs that warrants further investigation for the potential treatment of PDAC patients. 12 test samples in total [4 PDAC models (PDAC-1, PDAC-2, PDAC-3, PDAC-4; in three panel: primary human PDAC, primary tumor culture and mouse sample)] and reference sample (healthy control (mix/pool of healthy volunteers DNA) were analyzed as following (8 hybridizations); PDAC-1 primary human-Cy3 vs PDAC-4 cultured cells -Cy5 PDAC-1 cultured cells-Cy3 vs PDAC-4 mouse-Cy5 PDAC-1 mouse-Cy3 vs reference-Cy5 PDAC-2 primary human-Cy3 vs reference-Cy5 PDAC-2_cultured cells-Cy3 vs PDAC-2 mouse-Cy5 PDAC-3_primary human-Cy3 vs reference-Cy5 PDAC-3_cultured cells-Cy3 vs PDAC-3 mouse-Cy5 PDAC-4 primary human-Cy5 vs reference-Cy3 Normalized log2 ratio of (sample/references) data were calculated for 12 samples [*txt files on Series records]. Description of experimental/analysis design in r-program to generate 12 samples from 8 raw data files is provided in README.txt [Series supplementary file]
Project description:This project aims to compare gene expression in transgenic mice that overexpress TGFB1 to wild type mice. Transgenic mice have a fibrotic response in the atria, but not the ventricle. This is a 2 color design; dye swaps and a loop design were incorporated. Each group has N=4, with the exception being WT atria which has N=3 and was hybridied twice, the groups are Wt atria, WT ventricle, Transgenic atria, Transgenic ventricle. Comparisons of interest : Tg atria vs WT atria, Tg ventricle vs WT ventricle, WT atria vs.dWT ventricle, Tg atria vs Tg ventricle
Project description:In the heart, the serine carboxypeptidase cathepsin A (CatA) is distributed between lysosomes and the extracellular matrix (ECM). CatA-mediated degradation of extracellular peptides may contribute to ECM-remodeling and left ventricular (LV) dysfunction. This study aimed to evaluate the effects of CatA overexpression on LV remodeling. A proteomic analysis of the secretome of adult mouse cardiac fibroblasts upon digestion by CatA identified the extracellular antioxidant enzyme superoxide dismutase (EC-SOD) as a novel substrate of CatA (5-fold decreased abundance; p=0.0001). In vitro, cardiomyocytes and cardiac fibroblasts expressed and secreted CatA protein. EC-SOD protein was expressed and secreted only by cardiac fibroblasts. Cardiomyocyte-specific over-expression of CatA and increased activity in the LV of transgenic mice (CatA-TG) reduced EC-SOD protein levels by 43% (p<0.001). Loss of EC-SOD-mediated anti-oxidative protection resulted in accumulation of superoxide radicals (WT: 4.54±1.2 vs. CatA-TG: 8.62±2.3µmol/mg tissue/min; p=0.0012), increased inflammation, myocyte hypertrophy (WT: 19.8±1.0 vs. CatA-TG: 21.9±1.8µm; p=0.024), cellular apoptosis, and elevated mRNA expression of hypertrophy-related and pro-fibrotic marker genes, without effecting intracellular detoxifying proteins. In CatA-TG mice LV interstitial fibrosis formation was enhanced by 19% (p=0.028) and type I/type III collagen ratio was shifted towards higher abundance of collagen I fibers (p=0.026). Cardiac remodeling in CatA-TG was accompanied by increased LV weight/body weight and LV enddiastolic volume (WT: 50.8±5.8 vs. CatA-TG: 61.9±6.2 µl; p=0.018). Thus, in the heart CatA-mediated reduction of EC-SOD protein contributes to increased oxidative stress, myocyte hypertrophy, ECM remodeling and inflammation. This implicates CatA as a potential therapeutic target to prevent ventricular remodeling.
Project description:Investigating the transcriptional changes in mouse livers exposed to low and high fat diets for 11 weeks. Determine what gene changes in the Cyp1b1 null livers may be contributing to prevention of increased adiposity when givin a high fat diet. Three Comparison experiment. Comparing wild-type low fat vs Cyp1b1 null low fat, wild-type high fat vs Cyp1b1 null high fat and low fat vs high in wild-type mice. Sample size is 3 per group. Limma analysis data provided in Series supplementary file (Normalized log2 ratio of (Cy3/Cy5) representing test group/control group).