Identification of new markers of cutaneous melanoma invasion
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ABSTRACT: T1C3 is a clonal cell line from a human melanoma tumor, expressing PNA lectin, and characterized by a high capacity of dermal invasion. IC8 is a clonal cell line from a human melanoma tumor, negative for PNA lectin, and non-invasive. To identify new gene markers involved in the mechanisms of early invasion, oligonuclotide microarrays were used to assess transcriptional chnages between the two cell lines. Dye-swap hybridization were performed. Slides were scanned with a Genepix 4000 microarray scanner (Axon Instruments, Molecular devices, Sunnyvale, CA). For each hybridized spot, the Cy3 and Cy5 fluorescence values were obtained by using Genepix Pro 4.0 software (Axon Instruments) and were saved as a result file.
Project description:T1C3 is a clonal cell line from a human melanoma tumor, expressing PNA lectin, and characterized by a high capacity of dermal invasion. IC8 is a clonal cell line from a human melanoma tumor, negative for PNA lectin, and non-invasive. To identify new gene markers involved in the mechanisms of early invasion, oligonuclotide microarrays were used to assess transcriptional chnages between the two cell lines.
Project description:To characterize the consequences of the GATA3 silencing on the transcriptome of X-rays irradiated keratinocytes. Oligonuclotide microarrays were used to assess transcriptional changes over a time-course between 3 and 72h post-irradiation in a GATA3 knocked-down background (shGATA3) or in a normal background (ShSCR). Primary human keratinocytes were cultured in a semi-defined medium and then infected either with shGATA3 or with ShSCR lentiviral particles . Cells were then cultured 5 to 8 days up to confluence and then submitted to an 1 cGy dose of X-rays. Total RNA was extracted 4, 24, 48 or 72 hours after irradiation. After RNA amplification and labeling, gene profiling was performed using oligonucleotide microarrays (26 068 probes) to compare 1 cGy irradiated cells to sham-irradiated cells at individual times. Dye-swap hybridization were performed. Slides were scanned with a Genepix 4000 microarray scanner (Axon Instruments, Molecular devices, Sunnyvale, CA). For each hybridized spot, the Cy3 and Cy5 fluorescence values were obtained by using Genepix Pro 4.0 software (Axon Instruments) and were saved as a result file
Project description:To characterise the response of keratinocyte stem cells to ionizing radiation, gene profiling was performed using oligonucleotide microarrays (26068 probes). Cells were seeded in culture plates after cell sorting, irradiated the next day (2 Gy at 15 hours in culture), and RNA was prepared 3 hours after exposure. LOWESS normalisation was applied. dye-swap hybridization were performed. Slides were scanned with a Genepix 4000 microarray scanner (Axon Instruments, Molecular devices, Sunnyvale, CA). For each hybridized spot, the Cy3 and Cy5 fluorescence values were obtained by using Genepix Pro 4.0 software (Axon Instruments) and were saved as a result file
Project description:To characterize the global transcriptome of human keratinocyte stem cells (KSC) and keratinocyte progenitors (KP), primary basal keratinocyte subpopulations enriched in quiescent stem cells [Itg-?6bright / Trf-Rdim] or in cycling progenitors [Itg-?6bright / Trf-Rbright] were purified by flow cytometry from human skin samples. Sorted keratinocytes were then seeded in culture plates and maintained in culture medium overnight (15 hours), and genome-wide transcriptome analysis of these two subpopulations was directly performed, in order to globally maintain the initial phenotypes. After RNA preparation, gene profiling was performed using oligonucleotide microarrays (26068 probes). LOWESS normalisation was applied. Keywords: epidermis, keratinocyte stem cells, progenitors, transcriptome, stemness Dye-swap hybridization were performed. Slides were scanned with a Genepix 4000 microarray scanner (Axon Instruments, Molecular devices, Sunnyvale, CA). For each hybridized spot, the Cy3 and Cy5 fluorescence values were obtained by using Genepix Pro 4.0 software (Axon Instruments) and were saved as a result file
Project description:Analysis of four lung cancer cell lines transfected with a vector expressing the transcriptional repressor Snail versus a vector control. Aberrant Snail expression is known to induce an EMT program in lung cancers. Four lung cancer cell lines (H292, H358, H441, H1437) with stable overexpression of the human SNAI1 gene and vector expressing controls were collected at equal confluency with the miRNeasy Mini kit (Qiagen). One microgram of total RNA was labeled using miRCURY LNA™ microRNA Array Power Labeling kit by the UCLA Clinical Microarray Core. The labeled miRNAs were hybridized to Exiqon miRCURY LNA microRNA Array-6th Generation according to the manufacturer’s instructions. This array includes 927/648/351 human/mouse/rat miRNAs as well as 438 miRPlus miRNAs. The miRNA array slides were scanned using Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA) and processed by using the GenePix Pro 6.0 software (Axon Instruments). The raw data were normalized by using a combination of housekeeping miRNA, spike-in miRNA and invariant endogenous miRNAs.
Project description:C. jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human foodborne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion as evidenced by gentamicin-protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC demonstrated Cia secretion as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene as judged by B-galactosidase reporter assays and real-time RT-PCR. Microarray analysis revealed that DOC induced the expression of virulence genes (i.e., ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrate that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation to identify genes expressed by C. jejuni in response to in vivo-like culture conditions. Keywords: Stress response For the expression profiling arrays, an indirect comparison of gene expression was performed, where the expression profile of the C. jejuni F38011 cultured in the presence and absence of DOC was measured separately on different slides as described previously (26). Briefly, Cy5 labeled reference DNA from the C. jejuni F38011 strain was mixed with Cy3 labeled test cDNA (C. jejuni F38011 cultured in the presence or absence of DOC) and hybridized to the Campylobacter cDNA array (26) on separate slides. DNA microarrays were scanned using an Axon GenePix 4000B microarray laser scanner (Axon Instruments, Union City, CA) and the data for spot and background intensities were processed using the GenePix 4.0 software. To compensate for any effect of the amount of template and uneven Cy-dye incorporation, data normalization was performed as previously described (26). For the comparison of genes differentially expressed in the presence and absence of DOC,six hybridization measurements were generated per biological experiment (three technical replicate arrays and two replicate features per array).
Project description:Comparison of pre and post metamorphis phases of cane toad Keywords: Developmental Stages Samples were harvested at 9, 18, 28 (pre-metamorph), 30 and 53 (post-metamorph) days of age and RNA extracted using Trizol Reagent (Invitrogen). Total RNA (100 mg) was reverse transcribed (Qiagen) and labelled cDNA probes were generated using the fluorophores, Cy3 and Cy5 (Amersham Pharmacia Biotech). Prehybridization of slides, application of the probe to the microarray slides, hybridisation, and subsequent washing steps were performed according to manufacturer's instructions (Corning Microarray Technology). Slides were scanned and analysed with a GenePix 4000B laser scanner and GenePix 3.0 and 4.0 software (Axon Instruments).
Project description:Transcriptional analysis of wild-type V. cholerae isolated from the rabbit ileal loop was performed by quickly pelleting bacteria from the fluid accumulated in the ileal loops after 12 h of infection. The bacteria were then resuspended in Trizol reagent and frozen on dry ice. The wild-type bacteria from two independent experiments in two different rabbits were analyzed. RNA was isolated from the Trizol agent, treated with DNaseI (Ambion, Austin, Texas, United States), and cleaned by using the RNeasy kit (Qiagen, Valencia, California, United States). Labeling of cDNA and microarray hybridizations were performed as described [Yiliz et al. 2001, Mol. Micro. 53: 497-515]. Microarrays were scanned with a GenePix 400A instrument (Axon Instruments), using the GENEPIX 5.0 software. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
Project description:Late exponential growth phase (18 h) Emericella nidulans mycelia were transferred to minimal media also supplemented with 1.8 mM diamide, 75 mM H2O2 or 0.8 mM menadione (sodium bisulfite addition compound) and transcriptome profiles were recorded at 0.25, 0.5, 1, 3, 6 and 9 h incubation times. Double printed EST-based (2x4352 spots) DNA chips were used to monitor changes in cDNA populations prepared from mRNA pools isolated from oxidative stress exposed and untreated control cultures. Gene expression ratios were estimated with Genisphere (Hatfield, PA, USA) 3DNA Submicro EX Expression Array Detection Kit. Expression data were read with a GenePix 4000B microarray scanner (Axon Instruments). Intensity ratios were calculated with GenePix Pro 3.0 software. Note, processed data after LOESS-type normalization are presented on Series GSE2085 and all the signs were also reversed during data processing to reach proper log2(treated/control) channel intensity ratios. Keywords: time-course
Project description:Fly strain y[1]; bw[1]; e[4]; ci[1] ey[R] was obtained from the Bloomington Stock Center. Flies were reared in incubators at 25°C, 65% of relative humidity, and photoperiod of 12 hours. For gene expression analyses flies were exposed to BPA, DEHP, and high sugar for 48 hours (acute exposure). After exposure, they were flash-frozen in liquid nitrogen. Four replicas of each sample were collected and stored at -80°C. Total RNA was extracted from whole flies using TRIzol (Life Technologies). The synthesis of cDNA and its labeling with fluorescent dyes (Cy3 and Cy5) as well as hybridization reactions were carried out using 3DNA protocols and reagents (Genisphere). Slides were scanned using Axon 400B scanner (Axon Instruments) and GenePix Pro 6.0 software. Foreground fluorescence of dye intensities was normalized by the Loess method in Bioconductor / Limma. Dye "swaps," loop design.