Project description:To understand differences between resting and activated memory CD8+ T cells, we compared the global gene expression of ex vivo isolated naive and spleen and BM memory cells to in vitro activated spleen and BM memory cells. Single cell suspension from the spleen and bones of aged C57BL/6 mice were prepared. Naive (CD44-CD127+) and memory (CD44+CD127+) CD8+CD3+ T cells were then cytometrically sorted. Sorted cells were either immediately processed for RNA preparation or were activated with anti-CD3 and anti-CD28 for 42-44 hours. Total RNA was extracted using the NucleoSpin RNA (Macherey-Nagel). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 M-BM-5g total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to MG_U430_2 GeneChips (Affymetrix) in triplicates. Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.
Project description:We combined heritability analysis of larval development rate with a global expression analysis of this phenotype to investigate genotype by environment interactions across three ecologically relevant temperatures in the Glanville fritillary butterfly (Melitaea cinxia). We focused upon the development of final instar caterpillars which is greatly affected by temperature, and during this stage the caterpillars build up most of the resources for adult life. Second generation, lab reared larvae, initially collected from the M-CM-^Eland metapopulation, were reared in standard lab condition until 6th larval instar. At the beginning of the final (7th) instar stage the larvae were separated into of three temperature conditions: Cold treatment (temperature profile: 8M-BM-0C 18:00-9:59, 14M-BM-0C 10:00-11:59, 20M-BM-0C 12:00-15:59 and 14M-BM-0C 16:00-17:59) Standard treatment (temperature profile: 15M-BM-0C 17:00-6:59, 18M-BM-0C 7:00-8:59, 22M-BM-0C 9:00-10:59 and 26M-BM-0C 11:00-16:59) Hot treatment (temperature profile: 8M-BM-0C 20:00-7:59, 15M-BM-0C 8:00-9:59, 35M-BM-0C 10:00-17:59 and 15M-BM-0C 18:00-19:59) The temperature profiles mimic the diurnal thermal variation of the natural habitat (M-CM-^Eland islands) of samples. Cold treatment mimics a cool and cloudy summer, Standard represents an average temperature profile in the M-CM-^Eland islands and Hot treatment mimics an exceptionally hot and sunny summer, with cold night-time temperatures. The experiment contained several larval families (full-sib) of which three were selected for gene expression analysis. Samples were snap-frozen in liquid nitrogen during mid-development (after 6, 5 and 4 days, for Cold, Standard and Hot respectively). Additional larvae from the same treatments were assayed for survival and growth. Gene expression was analyzed using a mixed model approach to identify genes with potential heritable expression variation (variation among families), genes with plastic expression responses (treatment induced changes) and genes with treatment dependent expression that varies among families (family by treatment interactions). Full-sib larvae from three families (N170, N74 and O171) were exposed to three temperature treatments (Cold, Standard and Hot) during final (7th) instar stage. A total of 35 samples were used to analyze family, treatment and family by treatment interactions in gene expression, using a mixed model approach. This included 3 biological replicates in Cold and Hot treatments from each family and 5-6 biological replicates in Standard (5 in O171) per family. Techinal replicates from each family and additional techical replicates in family O171 (including dye swap replicates) were used to assess robustness of the findings.
Project description:Enterohemorrhagic Escherichia coli (EHEC) are transmitted from cattle to human by means of contaminated food products resulting from fecal contamination. Transcriptome analysis was performed to gain further insight into the metabolic pathways required for persistence and growth of EHEC in the bovine intestine. Understanding the physiology of EHEC in the gut of ruminants is critical to identifying the potential nutritional basis to limiting EHEC shedding. A global transcriptome analysis was performed to gain further insight into the metabolic pathways required for persistence and growth of EHEC in the bovine intestine. DNA microarrays were performed using RNA from EHEC O157:H7 EDL933 incubated in bovine small intestine content (BSIC) compared with cells incubated in M9-minimal media. Four biological replicates collected for bacterial cultures on separate days for each media and labelled following a dye-switch design : For each media two replicates labeled in Cy3 and two replicates in Cy5.
Project description:RNA-seq analysis of murine eGFP+ relbfl/flnfkb2fl/flCg1-Cre and Cg1-Cre splenic germinal center B cells identifies genes regulated by the transcription factors RELB and p52 (NF-kB2) in germinal center B cells. Germinal center B cells from 12-week old relbfl/flnfkb2fl/flCg1-Cre and Cg1-Cre littermate mice immunized with sheep red blood cells (SRBC) were isolated at day 7 after immunization by flow cytometric sorting from splenic mononuclear cells. RNA was isolated, amplified and submitted for RNA-sequencing on an Illumina HiSeq2500 instrument for 35-40 million 2x50 paired-ended reads.
Project description:We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridisation (aCGH). Gastric cancer tumor tissue samples and cell lines vs normal blood samples
Project description:Comparing the gene expression profiling of HDGF-silenced RD-ES cells and control RD-ES cells to identify genes regulated by HDGF in RD-ES cells. Keywords: expression analysis Control RD-ES cells and HDGF-silenced RD-ES cells were profiled on 22K Human Genome Array
Project description:Plasmodium falciparum parasites were treated with the PfPdx1 inhibitor 4PEHz and the transcriptional responses of three different timepoints throughout the intraerythrocyic developmental cycle of the parasite were compared to untreated parasites. The goal was to determine the functional consequences of attenuating vitamin B6 metabolism in the parasites using 4PEHz (4-phospho-D-erythronhydrazide) Two color array with reference pool design, at least 2 biological replicates from each timepoint of treated and untreated parasites.
Project description:The strain bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H] improved salt resistance of bdf1M-bM-^HM-^F. To gain further insight into the mechanism of BDF1 in suppressing bdf1M-bM-^HM-^F salt sensitivity, DNA microarray analysis was performed to determine the reason for the salt sensitivity of bdf1M-bM-^HM-^F cells and the process of how coexpression of SIR2 and BDF2 improves salt resistance. Transcriptomic analysis under salt treatment (0.6 mol.L-1 NaCl for 45 min) was performed using three different strains: bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H], bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242] and bdf1M-bM-^HM-^F[pRS316][pYX242]. The transcription of 3244 genes were significantly changed( > 2-fold) in bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H] compared with bdf1M-bM-^HM-^F[pRS316][pYX242] upon NaCl stress (0.6 mol.L-1 NaCl for 45 min). Only 281 genes were significantly changed ( > 2-fold) in bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242] compared with bdf1M-bM-^HM-^F[pRS316][pYX242] upon NaCl stress (0.6 molM-bM-^HM-^YL-1 NaCl for 45 min). BF2:bdf1M-bM-^HM-^F[pRS316][pYX242]. BF2B: bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242]. BF2S:bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H]. BF2B vs BF2 under salt treatment (0.6 mol.L-1 NaCl for 45 min); BF2S vs BF2 under salt treatment (0.6 mol.L-1 NaCl for 45 min).
Project description:Nectin-4 is a new therapeutic target in various carcinomas. We were interested in resistance to anti-nectin 4 ADC in breast cancer and the way to overcome it. Tumor DNA were extracted from sensitive and resistant pre-clinical models. Genomic profiles of samples were established by using array-CGH onto 4×180K CGH microarrays.
Project description:The processing of seafood for human or animal consumption creates vast amounts of by-product, which is often considered waste. Although some of these by-products are used as fishmeal, bone-meal or fertilizer, as a whole, it remains under utilized. Significant amounts of proteins, lipid fractions, vitamins, and other bioactive molecules are present in these by-products, all with potential beneficial properties that could be used as alternatives to fishmeal or as supplements for aquaculture species. In an attempt to investigate their potential benefit in Atlantic salmon fish nutrition, nine experimental diets were formulated using by-products originating from various seafood processing plants. A control basal diet (no by-product added) was also formulated. Juvenile Atlantic salmons were fed one of the nine experimental diets (30% marine by-product, 70% basal diet) or the basal diet and hepatic gene expression profiling was done on fish fed each diet after 14 and 56 days. Analysis of hepatic gene expression revealed a significant amount of differentially expressed genes for each diet with roles in various pathways and biological processes. By comparing differences in hepatic gene expression levels with the nutritional composition of the various feeds, we were able to identify a number of nutritional elements that affect specific gene families. This information will be very useful for the formulation of novel fish feeds, which may be designed with specific aims, such as rapid growth, increased immunity or better general health This specific study is aimed at evaluating the hepatic transcriptional responses in juvenile Atlantic salmon fed with fish feed formulation supplemented with one of nine marine by-products.