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: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.

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