Project description:Mammalian oogenesis is a complex mechanism entailing the maturation of primordial follicles into preovulatory follicles followed by the release of antral oocytes. In the ovary, the antral compartment consists of two populations of oocytes whose main difference regards the ability to resume meiosis and to develop, after the egg-sperm fusion, until the blastocyst stage. Still inexplicably, the antral oocytes distinguishable as Surrounded Nucleolus (SN; 70% of the antral oocytes population) are able to develop to the blastocyst stage, while those showing a Not Surrounded Nucleolus (NSN) arrest at the two-cell stage. RNA labeling and hybridization on microarray was done independently for SN and NSN oocytes with 2 replications. Two sets of 50 antral oocytes were collected in M2 medium and then were labelled with Cy3-CTP. Fluorescently labelled microarray targets were prepared using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent) with 2-round amplification. Cy5-CTP-labeled reference target was produced from mixture of Stratagene Universal Mouse Reference RNA and MC1 cell RNA. Targets were purified using an RNeasy Mini Kit (Qiagen), and then quantified on a NanoDrop scanning spectrophotometer (NanoDrop Technologies). Target cRNA was hybridized to the NIA Mouse 44K Microarray v3.0 (whole genome 60-mer oligo arrays, Agilent Technology, design ID 015087) according to manufacturers protocol (Two-Color Microarray-Based Gene Expression Analysis Protocol, Product # G4140-90050, Version 5.0.1). Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% and 10% PMT in both channels (except for the two oocyte Samples which were only scanned once each), with a scan resolution of 5um. All hybridizations compared one Cy3-CTP-labeled experimental target to the single Cy5-CTP-labeled reference target which was used for normalization. In the final analysis, we made a cutoff of 2.0 for the processed data (i.e., if value<2.0, it is set as 2.0) to minimize the noises from 2-round linear amplifications.

Project description:ratio experiment by combining dye-reversal ratio profiles CD4+ alpha beta T cells were positively sorted from LatY136F or from wild-type spleen using a MACS and anti-CD4 beads. Gamma delta T cells were sorted from the spleen of Lat3YF and of Eb-/- mice using MACS and anti-CD5 beads. In vitro differentiated Th1 and Th2 cells were generated by culturing naive wild-type CD4+ T cells for 4 days in complete RPMI in the presence of anti-CD3 (1 µg/ml) plus anti-CD28 (1 µg/ml) with the addition of IL-12 (10ng/ml) and of anti-IL-4 (5µg/ml) for Th1-polarizing conditions, and of IL-4 (20ng/ml) plus anti-IFN-gamma (10µg/ml) for Th2-polarizing conditions. RNA was prepared using the Trizol method followed by DNAse digestion. Microarray experiments were carried out as two-color ratio hybridizations. RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 4 µg total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with either cyanine 3-CTP (Cy3-CTP) or cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled antisense cRNA was precipitated over night with LiCl, ethanol washed and resuspended in water. The purified products were quantified at A552nm for Cy3-CTP and A650nm for Cy5-CTP and labeling efficiency was verified with a Nanodrop photometer (Kisker, Steinfurt, Germany). Before hybridization, 1.25 µg labeled cRNA of each product were fragmented and mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were done over night for approximately 17 h at 60°C. The slides were washed according to the manufacturer's manual and scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies). In order to compensate dye specific effects, and to ensure statistically relevant data, a color swap dye reversal was performed. Features were extracted with an image analysis tool Version A 6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmatics platform Resolver Built 4.0. Expression patterns were identified by stringent data analysis using anti-correlation of the dye reversal ratio profiles and a 2fold expression cut-off of the ratio experiments. By combining the first and the second criteria of analysis we filtered out data points with low P-value (P-value < 0.01), making the analysis robust and reproducible. Additionally, by using this strategy we did the data selection independent of error models implemented in the Rosetta Resolver system. Keywords: other

Project description:The microarrays experiments of three biological and one technical replicates were performed in YJR12 (wt) and YJR13 (myo1∆) strains. YJR12 (wild type) and YJR13 (myo1∆) strains were obtained as haploid segregants from a cross between YJR6 (myo1::HIS5 strain) and BY4741 (obtained from ATTC). Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base) with continuous shaking at 200 rpm. RNA was extracted from ribosomal pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies). The purity and integrity of the RNA was monitored using an Agilent Bioanalyzer (Agilent Technologies) following the manufacturer’s instructions. 1.0 µg of RNA extracted form ribosomal pellets from each sample was amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies). The amplified cRNA was labeled with 10mM Cyanine 5-CTP (Cy5) or Cyanine 3-CTP (Cy3) (Perkin Elmer Life Sciences). Labeled cRNA’s were purified with Qiagen RNeasy mini spin columns and dye incorporation was monitored on an Agilent Bioanalyzer. Hybridization of Cy5 and Cy3 labeled cRNA’s were performed using Yeast Oligo Microarray slides and hybridization kit from Agilent Technologies (Sheldon Manufacturing) at 60ºC for 17 hours. Slides were washed and scanned with a VersArray Chip Reader system (BioRad, Hercules, CA) at a resolution of 5mm with detector sensitivity values between 704-800 and laser power at 85%. Scanned images were transferred to the Imagene 3.0 software (Biodiscovery) for further analysis to locate spots, adjust the appropriate grid, and obtain the Cy3 and Cy5 TIFF files. The microarrays raw data generated with Imagene 3.0 were analyzed using Limma software (Bioconductor Package 1.7). The data was prepared for analysis by correcting for background intensity. The individual data sets were normalized using the locally weighted linear regression (Lowess) within each array. After normalization, the difference between the experimental and control signal was calculated, replicates were combined, and their averages were calculated. The fold change in gene expression was calculated by 2^(M), where M is the log2-fold change after background correction and normalization. An Empirical Bayes Statistics for differential expression analysis (eBayes statistics) was performed by Limma. Genes with a p-value ≤ 0.018 were established as a cutoff for differential expression. In addition, a false discovery rate (FDR) test was performed by Limma program.

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BAF250B-associated SWI/SNF chromatin-remodeling complex is required to maintain undifferentiated mouse ES cells

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