Project description:TGF-beta1 is a pleiotropic cytokine that regulates multiple cellular functions including cell proliferation, differentiation, and apoptosis. It has been identified as one of the mediators of renal hypertrophy and accumulation of extracellular matrix proteins in the diabetic glomerular mesangium. Previous results from our and other laboratories indicate that TGF-beta1 mediates some of the effects of high glucose and glucosamine on extracellular matrix gene expression in mesangial cells. The global regulatory mechanism(s), however, still remains to be understood. In this study, we have utilized an in vitro model of mouse mesangial cells (MES-13) to investigate the genetic effects of TGF-beta1 on global patterns of gene expression, transcriptome and signaling networks. A total number of 179 genes are regulated by TGF-beta1 in MES-13 cells at 2.0-fold, which cover across 44 physiological processes, 31 metabolic functions, and 11 major signaling pathways. We observed that several genes regulated by high glucose and glucosamine are also targeted by TGF-beta1 in MES-13 cells such as thioredoxin interacting protein, glutathione S-transferase theta 1, selenium binding protein 1, inhibitor of DNA binding 2, and plasminogen activator inhibitor-1. The results of this study further support the hypothesis that some effects of high glucose and glucosamine on mesangial gene expression are mediated by TGF-beta1. The identification of these common genes will help us to investigate further their roles in the development and progression of diabetic kidney disease. Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 Âµg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 Â°C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with 100 ng/ml TGF-beta1 for 24hrs. Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix M430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen, Valencia, CA). 15 mg of each cRNA was fragmented at 94 Â°C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100) containing a mixture of control cRNAs samples were heated at 94 Â°C for 5 min, equilibrated at 45 Â°C for 5 min, and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the master mix) were hybridized to GeneChip Mouse Genome 430 2.0 Array at 45 Â°C for 16 h in a rotisserie oven set at 60 rpm, then washed with non stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 Â°C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50 Â°C, stained with streptavidin-phycoerythrin (Molecular Probe), washed again with 6x saline/sodium phosphate/EDTA, stained with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin, and a third washing with 6x saline/sodium phosphate/EDTA using the GeneChip Fluidics Station 450. The arrays were then scanned using a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice. Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6.0 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (heep://appls.niaid.nih.gov/david).

Project description:The renal mesangial cells play an important role in the development of diabetic glomerulosclerosis and renal failure. We have previously demonstrated some of the effects of high glucose are mediated via the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 chips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on the transcriptomes of a mouse mesangial cell line (MES-13). Of the 34,000 genes on the chip, ~55-60% genes are detected in MES-13 cells. HG induces the expression of ~369 genes at >2-fold where 263 genes are up and 106 genes down regulated. Similarly, GlcN increases the expression of 120 genes and decreases 94 genes. Seventy-two genes are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The differential expressions of several genes found in the microarray are also confirmed by quantitative PCR. Significant pathways co-modulated by HG and GlcN are the thioredoxin system (a 20-fold increase in thioredoxin interacting protein expression), endoplasmic reticulum (ER) stress, extracellular matrix and interferon-inducible genes. Furthermore, HG and GlcN target various intracellular pathways including the mitogen-activated protein kinase, TOLL-like receptor, fructose and mannose metabolism, and the biosynthesis of steroids and N-glycans. We conclude from this microarray data and other experimental results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes ER stress, oxidative stress and the interferon-inducible gene expression to cause cell cycle arrest, ECM gene expression and apoptosis. Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with the desired concentrations of glucose and GlcN for 48h (LG, 5.5 mM; HG, 25 mM and GlcN, 1.5 mM + LG). Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix U430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA (from LG, HG and GlcN treated MES-13 cells) was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT) 24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled-cRNAs were purified in RNeasy spin columns (Qiagen, Valencia, CA). Fifteen mg of each cRNA was fragmented at 94 °C for 35 min in a fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). These cRNA fragments were then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100). The solution was equilibrated at 45 °C for 5 min and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the mater mix) were hybridized to GeneChip Mouse Genome U430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm. After the overnight hybridization, the chips were washed with a non-stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by a stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween-20) at 50 °C. The GeneChips were then stained with streptavidin-phycoerythrin (Molecular Probe), washed with 6x saline/sodium phosphate/EDTA, incubated with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin. Finally, a third wash with 6x saline/sodium phosphate/EDTA was performed using the GeneChip Fluidics Station 450. The arrays were then scanned in a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice. Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (http://appls.niaid.nih.gov/david). Keywords = glucose Keywords = glucosamine Keywords = mesangial cells Keywords = hexosamine Keywords: repeat sample

Project description:The renal mesangial cells play an important role in the development of diabetic glomerulosclerosis and renal failure. We have previously demonstrated some of the effects of high glucose are mediated via the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 chips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on the transcriptomes of a mouse mesangial cell line (MES-13). Of the 34,000 genes on the chip, ~55-60% genes are detected in MES-13 cells. HG induces the expression of ~369 genes at >2-fold where 263 genes are up and 106 genes down regulated. Similarly, GlcN increases the expression of 120 genes and decreases 94 genes. Seventy-two genes are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The differential expressions of several genes found in the microarray are also confirmed by quantitative PCR. Significant pathways co-modulated by HG and GlcN are the thioredoxin system (a 20-fold increase in thioredoxin interacting protein expression), endoplasmic reticulum (ER) stress, extracellular matrix and interferon-inducible genes. Furthermore, HG and GlcN target various intracellular pathways including the mitogen-activated protein kinase, TOLL-like receptor, fructose and mannose metabolism, and the biosynthesis of steroids and N-glycans. We conclude from this microarray data and other experimental results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes ER stress, oxidative stress and the interferon-inducible gene expression to cause cell cycle arrest, ECM gene expression and apoptosis. Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with the desired concentrations of glucose and GlcN for 48h (LG, 5.5 mM; HG, 25 mM and GlcN, 1.5 mM + LG). Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix U430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA (from LG, HG and GlcN treated MES-13 cells) was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT) 24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled-cRNAs were purified in RNeasy spin columns (Qiagen, Valencia, CA). Fifteen mg of each cRNA was fragmented at 94 °C for 35 min in a fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). These cRNA fragments were then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100). The solution was equilibrated at 45 °C for 5 min and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the mater mix) were hybridized to GeneChip Mouse Genome U430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm. After the overnight hybridization, the chips were washed with a non-stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by a stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween-20) at 50 °C. The GeneChips were then stained with streptavidin-phycoerythrin (Molecular Probe), washed with 6x saline/sodium phosphate/EDTA, incubated with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin. Finally, a third wash with 6x saline/sodium phosphate/EDTA was performed using the GeneChip Fluidics Station 450. The arrays were then scanned in a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice. Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (http://appls.niaid.nih.gov/david).

Project description:Goal of experiment: Identification of differentially expressed immune genes from male and female BWF1 lupus-prone mice. (Female incidence is higher than male--attempting to find sex hormone regulated genes that may contribute to this difference). Whole spleen was taken from pre-lupus (4 months old) BWF1 (females are lupus-prone) male and female mice. Preparation of cDNA. Double-stranded cDNA was synthesized from purified RNA. The first strand was synthesized by incubating 5 μg of RNA with 100 pg/ml T7-(dT)24 primer (HPLC purified DNA primer sequence: 5’-GGCCAGTGAATTGTAATACG ACTCACTATAGGGAGGCGG-(dT)24 -3’ Genset Corp, San Diego, CA) at 70°C for 10 minutes. Samples were incubated for 1 hour at 42°C with the following mix: 1X first strand buffer, 10 mM dithiothreitol, 500 μM each dNTP, 200 U SuperScript II in diethylpyrocarbonate (DEPC)-treated water up to 20 μl. Second strand synthesis was performed by incubating the first strand with the following mix for 2 hours at 16°C: 1X second strand reaction buffer, 200 μM dntps, 10 U E. coli DNA ligase, 40 U E coli DNA Polymerase I, 2 U of E. coli RNase H up to 150 μl with DEPC-treated water (all reagents were contained in SuperScript Choice System for cDNA Synthesis, Invitrogen). A phenol/chloroform extraction was performed on the ds-cDNA preparation before biotin-labeled cRNA was generated. Synthesis and fragmentation of biotin-labeled cRNA (in vitro transcription). The ENZO BioArrayTM HighYieldTM RNA Transcript Labeling Kit (T7) (Enzo diagnostics, Inc., Farmingdale, NY) was used to produce large amounts of hybridizable biotin-labeled RNA targets by in vitro transcription from the ds-cDNA. The following mix was incubated at 37°C for 5 hours: 1 μg of ds-cDNA, 1X HY reaction buffer, 1X biotin labeled ribonucleotides, 1X dithiothreitol, 1X T7 RNA Polymerase. Biotin-labeled cRNA was run over RNeasy spin columns (Qiagen), quantified, and run on an agarose gel to visualize the size distribution of labeled transcripts. Twenty micrograms of cRNA was incubated with 1X fragmentation buffer for 35 minutes at 94°C. (5X fragmentation buffer: 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc). After fragmentation, the samples were stored at -20°C until the hybridization was performed. Sample hybridization. Oligonucleotide microarrays (MGU74v2 A, B, and C GeneChip probe arrays; Affymetrix) were hybridized with labeled cRNA derived from spleens from individual mice. For each array,15 μg of fragmented cRNA was mixed with a hybridization cocktail consisting of 1X hybridization buffer (2X hybridization buffer: 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween), 0.5 mg/ml acetylated BSA (Invitrogen), 0.1 mg/ml herring sperm DNA (Promega), and water (BioWhittaker) up to 300 μl). Biotin labeled cRNA transcripts of the E. coli and P1 bacteriophage genes, BioB, bioC, bioD, and cre (GeneChip Eukaryotic Hybridization control kit, Affymetrix) were spiked into each hybridization mix at 1.5, 5, 25, and 100 pM to evaluate sample hybridization efficiency for each array. The hybridization cocktail was heated to 99°C and then 45°C for 5 minutes each before it was centrifuged to remove any insoluble material. The array was equilibrated to room temperature, moistened with 1X hybridization buffer, and incubated for 10 minutes at 45°C with rotation. After incubation, the buffer solution was removed from the array. The array was filled with 300 μl of the hybridization cocktail, placed in a rotisserie box in a 45°C oven, and incubated for 16 hours while rotating at 60 rpm. Washing and staining of array. The hybridization cocktail was removed and the GeneChip Fluidics Station 400 (Affymetrix) with Microarray Suite software (Affymetrix) was used to wash and stain the probe arrays with the following protocol: 10 cycles of 2 mixes/cycle with wash buffer A at 25°C, 4 cycles of 15 mixes/cycle with wash buffer B at 50°C, 30 minute incubation with staining solution at 25°C, 10 cycles of 4 mixes/cycle with wash buffer A at 25°C. Wash buffer A -- non-stringent wash buffer (6X sodium chloride sodium phosphate + ethylenediaminetetraacetic acid (SSPE), 0.01% Tween-20). (20X SSPE: 3 M NaCl, 0.2 M NaH2PO4, 0.02 EDTA) (BioWhittaker). Wash buffer B – stringent wash buffer (100mM MES, 0.1 M [Na+], 0.1% Tween 20). Staining solution (1X 2-(N-Morpholino)ethanesulfonic Acid (MES) stain buffer, 2 mg/ml acetylated BSA, 10 μg/ml Streptavidin Phycoerythrin (SAPE), and water up to 600 μl). (12X MES stain buffer: 1.22 M MES, 0.89 M [Na+]). Analysis. After staining, the probe arrays were scanned using the GeneChip 3000 Scanner (Affymetrix) with Microarray Suite software (Affymetrix). Technical and assay variation between arrays was corrected for by multiplying or dividing the overall intensity of each array by a scaling factor so that the overall intensity of each array was equivalent to facilitate comparison analysis. Keywords = BWF1 total spleen Keywords: repeat sample

Project description:Mesangial cells (MC) respond to various insults, such as hyperglycemia, with expressive phenotypic changes. Modifications in the patterns of MC proliferation, death, and the balance between matrix production and degradation affect directly glomerular function and ultimately lead to glomerulosclerosis. MC activation by growth factors (GF) has been demonstrated to be a trigger to changes in cell phenotype, and blockade of specific systems tend to attenuate glomerular damage. The identification of elements activated following GF stimulation of MC may facilitate the comprehension of the pathophysiology of glomerular diseases. By means of DNA microarray technology we identified that three inhibitors of DNA binding (ID1, ID3, and ID4) were among the most significantly regulated genes in serum-treated MC. We extended these studies and demonstrated that ID gene mRNA expression is markedly and rapidly affected by the pro-fibrotic cytokine TGF-beta1. Of interest, bone-morphogenetic proteins (BMPs), members of the TGF-beta superfamily with anti-fibrotic effects, also enhanced the expression of ID genes. Additional experiments indicate that these genes, classically described as dominant negative inhibitors of E-proteins, are regulated by TGF-beta1 and BMPs through distinct pathways and may act as immediate-early genes. Our results suggest that ID genes may play an active role in MC fate determination through undefined mechanisms. Keywords: comparative gene regulation by serum treatment The GE HealthCare CodeLink Human UniSet I Bioarray system, containing 10 000 gene probes, was used to generate global transcription profiles of human Mesangial Cells. All reagents were provided in the CodeLink Expression Assay Kit (GE HEALTHCARE, Piscataway, NJ, USA), except where noted. cRNA synthesis was performed according to the manufacturerâs instructions from 10 Âµg of pooled total RNA, purified on RNeasy columns (QIAGEN GmbH, Hilden, Germany), and quantified by UV spectrophotometry. cRNA was fragmented by heating at 94Â°C for 20 min in the presence of magnesium and hybridized overnight at 37Â°C in an appropriate buffer to a CodeLink slide under continuous shaking in an Innova 4080 incubator (New Brunswick, Edison, USA) at 300 rpm. After the hybridization, arrays were washed in 0.75x TNT (1x TNT: 100 mM Tris-HCl pH 7.6, 150 mM NaCl, and 0.05% Tween 20) at 46Â°C for 1 h followed by incubation with streptavidin-Cy5 (GE HealthCare) at room temperature for 30 min in the dark. Arrays were then washed in 1x TNT twice for 5 min and rinsed in 0.05% Tween-20. The slides were dried by centrifugation and kept in the dark until scanning. Images were captured on an GenePix 4000B Scanner (AXON INSTRUMENTS, Arlington, TX) using CodeLink Expression Scanning Software and were analyzed using CodeLink Expression Analysis Software (GE HEALTHCARE). This software generates normalized intensity values for each gene in each microarray, using the overall mean intensity of the array as the normalization standard.

Project description:STUDY THE EFFECT OF TGF beta ON SCHISTOSOME ADULT WORMSGoal: Primary: Assess the effects of TGF beta on transcription of adult male and female schistosomes. Secondary: Identify the pathways regulated by TGF beta in worm pairs, mature males, mature females, immature males and immature females.METHODS:Parasite: Schistosoma mansoni employed is the NMRI strain that is maintained in a Puerto Rican Biomphalaria glabrata and Golden Syrian hamsters. The life cycle is maintained in the LoVerde lab.Cercariae obtained from infected snails were used to infect experimental animals. The cercariae wre shed from snails exposed to multiple miracidia for mixed infections or from snails exposed to a single miracidia for single-sex infections. Schistosome worms were recovered from hamsters by perfusion (Duvall and DeWitt, 1967) 45 days post infection. Freshly perfused adult worms containing mixed-sex or single-sex infections were collected from infected golden hamsters, washed and incubated for overnight at 37C, 5% CO2 in MEM medium supplemented with 1 mM sodium pyruvate, 1 X non-essential amino acid solution, 1 X amino acid solution, 100 U ml penicillin, 100 ug ml streptomycin 1 ug ml amphotericin B (antibiotic antimycotic solution) and 2 mM Glutamax-I. Recombinant human TGF- beta1 was added individually to about 25 pairs or 30 individual schistosomes to a final concentrations of 1.0 nM and incubation was continued for another 24 hr. The controls were treated as above except no TGF-beta1 was added. The worms were placed in RNAlater and sent to WTSI (Anna) for RNA extraction and sequencing.Duvall RH, Dewitt WD. An improved perfusion technique for recovering adult schistosomes from laboratory animals. Am J Trop Med Hyg 1967;16:483-486.. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

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Transcriptome profiling of Transforming Growth Factor B1-Induced Mesangial Gene Expression

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