Project description:B-cell chronic lymphocytic leukemia (B-CLL) shows an incredibly high heterogeneity in the clinical course, spanning from rapidly aggressive to completely indolent behaviour. In order to identify the correspondent gene expression variability, we investigated 29 cases of untreated B-CLL using microarray. Firstly, two robust CLL clusters were identified applying multiple unsupervised clustering algorithms. Their separation was mainly determined by the differential expression of several genes included into two gene groups designated OxPhos and Lyn clusters. OxPhos gene cluster, previously identified in a subset of diffuse large B-cell lymphomas, comprises genes coding for some respiratory chain enzymes, ribosomal proteins and translation factors. Moreover, increased levels of genes involved in the regulation of apoptosis and in the proteasome-ubiquitin complex and the down-regulation of LYN gene, member of B-cell receptor pathway, characterized the OxPhos CLL subset. These B-CLL biological clusters did not reveal any preferential distribution of Ig mutated or Ig unmutated CLL prognostic groups. Furthermore, we applied another unsupervised algorithm (Subtractive Unsupervised Analysis) after the exclusion of genes characterizing the previously identified CLL subsets. At this point, we could identify two new CLL clusters, showing a clear association to the Ig mutational status. In addition to the ZAP-70 and LPL genes, the patients with unmutated Ig expressed higher level of some interesting genes involved in B cell activation, cell cycle regulation, apoptosis resistance and angiogenesis. In conclusion, we showed that B-CLL is characterized by an intrinsic heterogeneity in gene expression pattern, which overcomes the influence of the immunoglobulin mutational status on B-cell chronic lymphocytic leukemia profiles. SUBMITTER_CITATION: abstract in Blood, Volume 108, Issue 11 [2780] Angiopoietin-2 Expression in B-Cell Chronic Lymphocytic Leukemia: Association with Clinical Outcome and Immunoglobulin Heavy-Chain Mutational Status. "Rossana Maffei, Silvia Martinelli, Ilaria Castelli, Rita Santachiara, Elena Morandi, Patrizia Zucchini, Marcella Fontana, Anna Maria Colacci, Roberto Serra, Roberto Marasca, Giuseppe Torelli - Oncology and Hematology, University of Modena and Reggio Emilia, Modena, Italy; Excellence Environmental Carcinogenesis, ARPA, Bologna, Italy; Social, Cognitive and Quantitative Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Ita Experiment Overall Design: Twenty-nine B-CLL patients, who have attended the haematology clinic of Modena Hospital, were enrolled in this study. Total RNA from mononuclear cells was isolated using RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturerâ??s instructions For all 29 specimens, fluorescently-labelled cRNA was generated by in vitro transcription using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo alto, CA, USA) according to the manufacturerâ??s instructions. Amplified cRNA of each patient was labelled with cyanine 5-CTP (Perkin-Elmer, NEN Life Science, Boston, MA, USA) in each experiment. Mix of equal amount of total RNAs from our cohort was used as reference control in all microarray gene-profiling experiments. Amplified reference cRNA was labelled with cyanine 3-CTP (Perkin-Elmer, NEN Life Science, Boston, MA, USA) in each experiment. Moreover, cRNA products were purified using RNeasy columns (Qiagen). 1 ï?g of Cy5-labelled cRNA was mixed with the same amount of Cy3-labelled reference cRNA and then mixed cRNAs were fragmented to an average size of ï?¾50-100 nt by incubation at 60Â°C for 30 min using in situ Hybridization kit-plus (Agilent). Samples were hybridized on Agilent Human 1A Oligo Microarray, ink-jet printed microarray, comprising 20,173 (60-mer) experimentally validated oligonucleotide probes (features).33 After hybridization for 17 h at 60Â°C, slides were washed according to Agilent SSC protocol instructions and then scanned using a confocal laser scanner (Agilent Technologies). Experiment Overall Design: Fluorescence data were analysed with Feature Extraction Software v.7.5 (Agilent Technologies). Log10 ratio of the dye-normalized Cy3 and Cy5 channel signals were calculated, then a p-value and a final error were obtained to establish the significance of each feature.

Project description:In this study, we employ high-density oligonucleotide microarrays to characterize the MutaMouse FE1 cell line at various stages of cell growth, in primary MutaMouse lung epithelial cell cultures, and in whole lung. Global transcriptional analysis and real-time RT-PCR was applied to (1) further define the cellular origin of the FE1 cell line and its responses under different culture conditions (media and substratum), (2) provide insight into the transcriptional differences in cellular processes between FE1 cultures compared to whole lung tissues, more specifically in toxicological response, and (3) preliminarily examine FE1 culture response to exposure of benzo(a)pyrene compared to whole animals. Total RNA samples from 3 cell culture types (50% FE1, %100 FE1, and Primary lung) or MutaMouse lung were labeled with Cyanine 5-CTP, and universal reference total RNA (Stratagene, CA, USA) was labeled with Cyanine 3-CTP (Perkin Elmer Life Sciences, Woodbridge, ON, Canada) using Agilent linear Amplification kits (Agilent Tech. Inc. Mississauga, ON, Canada) following the manufacturer's instruction. Briefly, double-stranded cDNA was synthesized using MMLV-RT with T7 promoter primer, starting with 5 ug total RNA. Cyanine-labeled cRNA targets were in vitro transcribed using T7 RNA polymerase. The synthesized cRNA was precipitated by LiCl and fragmented at 60 degrees C for 30 min with fragmentation solution. Cy5- sample cRNA and Cy3- reference cRNA were hybridized to Agilent mouse development microarrays (containing ~20,000 unique 60 mer oligonucleotides; Agilent Tech. Inc. Mississauga, ON, Canada) at 60 degrees C overnight with Agilent hybridization solution and washed according to manufacturer's instruction. Arrays were scanned on a VersArray ChipReader (BioRad Laboratories Ltd., Waterloo, Ontario, Canada), and data were acquired with ImaGene 5.5 (BioDiscovery, Inc. CA, USA). Present calls were determined as signals that were greater than the mean plus three times the standard deviation of the average of the negative control spots.

Project description:Peumonia is the most common cause of death due to infectious diseases in the western hemisphere. The molecular events associated with pulmonary infections caused by Streptococcus pneumoniae and Influenza A virus are incompletely understood. Pathophysiological and protective processes are initiated by immune receptors specifically recognizing pathogenic structures serving to elicit a qualitatively and quantitatively adequate immune response. To provide a molecular framework towards a better understanding of the processes relevant in severe infectious pneumonia we performed a transcriptome analysis of lungs from mice infected with S. pneumoniae or Influenza A virus. Overall, we detected 1300 genes that exhibit significant differential expression after infection with either pathogen. Of those were approximately 36 % specific for pneumococcal and 30 % specific for the viral infection, yielding pathogen-specific as well as common inflammatory transcriptional signatures. Characteristically, these results resolve not only a differential response on the cytokine and chemokine level, although common induction of type I and type II interferons, TNFa, and IL-6 underlies both infections, but emphasize the potentially important role exerted by many genes implicated in the regulation and fine tuning of the inflammatory response. Furthermore, we noted a specific decrease in B cells after S. pneumoniae infection, which is not solely confined to the lung. Massive induction of apoptosis in pulmonary B cells could reflect a pneumococcal virulence strategy aiming against the lymphocyte population that is of utmost importance for the defence against capsulated bacteria. The pathophysiological consequences of Influenza A virus infection become obvious through differential induction of genes implicated in tissue regeneration and proliferation associated with detection of the emerging protective T cell response. These results may provide new insights into the pathogenesis and protective mechanisms important for the development of improved diagnostic and therapeutic strategies. Microarray analyses were performed as two-colour hybridizations. RNA was labelled with a Fluorescent Linear Amplification Kit (Agilent Technologies). Briefly, 4 Âµg total RNA was reverse transcribed 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. Purified products were quantified at A552nm for Cy3-CTP and A650nm for Cy5-CTP. Prior hybridization, 1.25 Âµg labelled cRNA each were fragmented, mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were performed for 17 h at 60Â°C. The slides were washed according to the manufacturer's protocol and scanning of the arrays was performed with 5 Âµm resolution using a DNA microarray laser scanner (Agilent Technologies). To compensate for dye specific effects, and to ensure validity of the data, a colour swap analysis (fluorescence reversal) was performed. Features were extracted with the image analysis tool Version A6.1.1.1 from Agilent Technologies. Subsequent data analyses were carried out on the Rosetta Inpharmatics platform Resolver Built 3.2.2. Differential expression patterns were identified by applying 2-fold expression level cut-offs and an anti-correlation of the dye reversal experiments with an error weighted p-value < 0.05.

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

Dataset Information

Transcription profiling of human motor cortex in sporadic amyotrophic lateral sclerosis

Publications

Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets