Project description:The purpose of the McrBC methylation microarray* assay is to determine which regions of a genome are methylated versus those that are unmethylated in a single Arabidopsis thanliana genotype. McrBC is a methylation-sensitive enzyme that restricts DNA only at purine-Cmethyl half sites when separated between* 50bp and 3kb. A designated amount of DNA from a particular genotype is sheared to a size range of 1kb-10kb using nebulization. We restrict half of the nebulized DNA with McrBC, and the methylated fraction is then removed from the unmethylated fraction through gel purification of DNA fragments greater than 1kb.* The remaining nebulized DNA is subjected to the same gel purification scheme, but with no McrBC treatment. In a single hybridization, the untreated sample is labeled with Cy5 and the McrBC-treated sample with Cy3.* Thus, after labeling and microarray hybridization, the ratio of normalized Cy5 to normalized Cy3 represents the relative methylation at the sequence represented by the spot on the microarray.* Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. The two samples in this series are complementary hybridizations in a dye-swap analysis. wild-type seedlings, 9 days old. Below is the normalized result of the paired dye swap samples VC134 and VC136. The ANOVA model of Kerr, Martin and Churchill (2000) was used to analyze the data from the dye-swap experiments, with terms included to account for gene, dye-by-gene, treatment-by-gene, and random error terms. The style of hypothesis test proposed by Black and Doerge (2002) was applied to each of the features represented on each array, with rejection of the null hypothesis indicating a significant change in fluorescence intensity. To account for the number of hypothesis tests being made, and thus provide some level of error rate control, significance was assessed using both family-wise error rate (FWER) and false discovery rate (FDR) controlling methods. The step-down multiple comparisons procedure of Holm (1979) was used to control the FWER below alpha = 0.01, while the step-up procedure of Benjamini and Hochberg (1995) was used to control the FDR below alpha = 0.01. For the purposes of this experiment, the hypotheses were assumed to be independent. Features found to be significantly enriched for DNA methylation after hypothesis-testing with a controlled error rate are flagged in the last two columns. Keywords: other

Project description:The purpose of the McrBC methylation microarray assay is to determine which regions of a genome are methylated versus those that are unmethylated in a single Arabidopsis thaliana genotype. McrBC is a methylation-sensitive enzyme that restricts DNA only at purine-Cmethyl half sites when separated between 50bp and 3kb. A designated amount of DNA from a particular genotype is sheared to a size range of 1kb-10kb using nebulization. We restrict half of the nebulized DNA with McrBC, and the methylated fraction is then removed from the unmethylated fraction through gel purification of DNA fragments greater than 1kb.* The remaining nebulized DNA is subjected to the same gel purification scheme, but with no McrBC treatment. In a single hybridization, the untreated sample is labeled with Cy5 and the McrBC-treated sample with Cy3. Thus, after labeling and microarray hybridization, the ratio of normalized Cy5 to normalized Cy3 represents the relative methylation at the sequence represented by the spot on the microarray. Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. ddm1 seedlings, 9 days old. The two samples in this series are complementary hybridizations in a dye-swap analysis. This is the normalized result of the paired dye swap samples VC131 and VC135. The ANOVA model of Kerr, Martin and Churchill (2000) was used to analyze the data from the dye-swap experiments, with terms included to account for gene, dye-by-gene, treatment-by-gene, and random error terms. The style of hypothesis test proposed by Black and Doerge (2002) was applied to each of the features represented on each array, with rejection of the null hypothesis indicating a significant change in fluorescence intensity. To account for the number of hypothesis tests being made, and thus provide some level of error rate control, significance was assessed using both family-wise error rate (FWER) and false discovery rate (FDR) controlling methods. The step-down multiple comparisons procedure of Holm (1979) was used to control the FWER below alpha = 0.01, while the step-up procedure of Benjamini and Hochberg (1995) was used to control the FDR below alpha = 0.01. For the purposes of this experiment, the hypotheses were assumed to be independent. Features found to be significantly enriched for DNA methylation after hypothesis-testing with a controlled error rate are flagged in the last two columns. Keywords: other

Project description:A set of 5,908 69-70 mers oligonucleotides that correspond to all C. glabrata putative open reading frames and some non-coding sequences was designed and synthesized at Pasteur Institute, France. Each oilgonucleotide was dissolved into 50% DMSO and spotted on UltraGAPsTM glass slides (Corning Incorporated, Acton, MA) in duplicate to generate whole genome microarray for C. glabrata. The microarray was printed at Biotechnology Research Institute/National Research Council of Canada in Montreal. The sequence information of the oligonucleotides and the micorarray setup can be found at platform GPL3922. Using the microarray, the transcriptional profiles of C. glabrata cells that were coincubated with J774A.1 murine Macrophage-like cells for 2 and 6 hrs were examined. At each time point, three biological repeats were done. 20 ug of total RNA from either macrophage-ingested C. glabrata cells (experimental sample) or the medium-alone controls (control sample) was used to synthesize Cy5- or Cy3-labeled cDNA probes with an anchor primer [(dT)18(dA/dG/dC)] in the presence of Cy5- or Cy3-dCTP (PerkinElmer). For each pair of experimental and control samples, dye-swap was performed, that is, two probe mixtures, the Cy5-experimental plus the Cy3-control probes, and conversely, the Cy3-experimental plus the Cy5-control probes were individually hybridized with a single C. glabrata microarray slide. The hybridization and subsequent washing conditions follows the instruction on the manual for UltraGAPsTM glass slides. The hybridized microarrays were scanned with Axon 4000B scanner, and Data were extracted using Axon GenePix program. Log2 (experimental/control) for each spot on the microarray was calculated. From each dye-swap experiment, the Log2 (experimental / control) for each spot were averaged. Finally, 6 Log2 (experimental/control) values (3 repeats × duplicate spots on each slide) were imported into SAM (significance analysis of microarrays) software for statistical analysis. For genes that are considered significantly induced or repressed, the median of false discovery rate (FDR) equals to 0, while the 90% FDR is less than 0.001. Keywords: transcriptional profiling by microarray

Project description:In the present study we investigate the effect of maternal pulmonary exposure to titanium dioxide (UV-Titan designed for use in the paint and lacquer industry) on prenatally exposed offspring. Time-mated mice (C57BL/6BomTac) were exposed by inhalation for 1 h/day to 42 mg UV-titan/m3 aerosolized powder or filtered air during gestation days (GD) 8-18. We evaluated levels of DNA strand breaks using the comet assay in bronchioalveolar lavage (BAL) cells and liver cells of the time-mated mice, and in liver cells of the offspring. In parallel, we analyzed changes in gene expression in the liver tissue of offspring using DNA microarrays. We demonstrate that UV-Titan did not induce DNA strand breaks in the time-mated mice or their offspring. Transcriptional profiling of newborn liver tissue revealed changes in the expression of genes related to the retinoic acid (RA) signalling pathway in the females, while gene expression in male offspring was relatively unaffected by exposure. Time-mated, nulliparous mice (C57BL/6BomTac, Taconic Europe, Ejby, Denmark) were exposed to filtered clean air or approximately 42 mg/m3 UV-Titan on GD 8M-bM-^@M-^S18 for 1 hr/day by a whole-body exposure.Delivery was expected on GD 20, and designated postnatal day (PND) 0. On PND 2, one offspring (M-bM-^@M-^\newbornM-bM-^@M-^]) per litter was killed by decapitation, to yield litters with at least 5 offspring per exposure group. The newborn offspring were collected two days after birth. On PND 23-24 (M-bM-^@M-^\at weaningM-bM-^@M-^]) one male and one female per litter were killed. Organs were dissected, weighed, placed in NUNC cryotubes, snap frozen in liquid N2 and stored at -80M-BM-0C until analysis. Exposed dams were killed on PND 24-25 (26-27 days after last exposure). DNA strad break detection was carried out in bronchoalveolar lavage fluid in dams by COMET assay. Total RNA was extracted from offspring livers and gene expression profiling was carried out on whole liver tissues from offspring. 9 UV-Titan treated and 7 controls mice consisting of 8 males and 8 females mRNA, hepatic, developmental, Toxicogenomics, nano-titanium dioxide

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Screening for candidate clock-controlled genes

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