Project description:Surgically resected gastric adenocarcinoma samples and their paired disease-free (R0) marginal nonmalignant tissue were obtained from 14 patients with gastric adenocarcinoma. RNA was isolated using the RNAeasy Kit (Qiagen Valencia, CA) from 15 mg of tissue. The quality of total RNA and its integrity was assessed using the Bioanalyzer 2100 (Agilent, Palo Alto, CA) and RIN value (RNA Integrity Number) was recorded for all the samples for intact 18S and 28S rRNA. Total RNA (800 ng) from each sample was reverse transcribed and linear amplified using the low RNA input linear amplification kit (Agilent Technologies). After synthesis of the first and second strand of cDNA, the product was used in an in vitro transcription reaction to generate cRNAs in the presence of cyanine 3-labeled UTP (Cy3) for normal and cyanine 5-labeled UTP (Cy5) for tumor. Fragmented Cy3-labeled cRNA of the control sample were mixed with equal amounts of Cy5-labeled cRNA from the gastric tumor sample, and the mixtures were hybridized onto 44K whole human genome DNA microarrays (Agilent Technologies) for 17 hrs at 65M-BM-0C with constant rotation (10 rpm). Subsequently, the arrays were washed according to manufacturerM-bM-^@M-^Ys instructions. The slides were scanned using an Agilent microarray scanner (G2565AA), and the images were processed using the Agilent feature extraction software AFE 9.5. GeneSpring GX v10.1 (Agilent Technologies) was used to analyze the expression profiles obtained after microarray hybridization. The feature extraction files from all the samples were uploaded to GeneSpring. Following intensity-dependent normalization (Lowess), the data was subjected to statistical analysis. A two tailed studentM-bM-^@M-^Ys t-test was used to determine significance of the differences observed between the normal and tumor samples. Benjamini Hochberg algorithm was used to compute false discovery rates. Genes were filtered based on P value threshold of 0.001 and false discovery rate of less than 1%. Transcriptional profiling of normal vs. tumor tissues from 14 patients with gastric cancer. Two-color experiment.

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.

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

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Gene expression profiling of gastric cancer

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