Expression microarray analysis of acquired gemcitabine resistance
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE2352 | GEO | 2005/12/21
SECONDARY ACCESSION(S): PRJNA91645
REPOSITORIES: GEO
Similar Datasets
Project description:This series repressents the data set from the paper "Expression microarray analysis and oligo array CGH of aquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations". Biological material: One solid mouse tumor "Colon 26A" was routinely maintained by successive transplantation. A subset of mice with this tumor was treated at the maximum tolerable dosage of Gemcitabine and successively transplanted 5 times and similarly treated. The last passage was completly resistant and designated, "Colon 26G". Expression array: Total RNA was isolated from frozen mouse tumors using Tryzol. Single stranded cDNA was synthetized from 30 mg of total RNA by reverse transcription using aminoallyl labeled dUTP (Ambion). Labelling was performed according to the aminoallyl labeling protocol. Briefly, cDNA was incubated at room temperature for 1 h with fluorolink monofunctional cy5 or cy3 (Amersham) followed by 15 min 4 M hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen) and mixed with 12 mg poly(dA) (Amersham), 60 mg yeast tRNA (Sigma) and 24 mg Cot-1 DNA (Invitroen). The probe was dissolved in 127 ml hybridization mixture containing 46% formamide, 9.5% dextran sulfate, 2x SSC and 0.2% SDS. The probe was heated to 70°C for 10 min and annealed at 37°C for 1 h. Slides were hybridized for 14 h at 37°C over-night (HyArray 12â¢, Perkin Elmer). After hybridization the slides were washed in the HybArray 12⢠with 50% formamide, 2x SSC, pH 7 at 35°C for 15 min, followed b PI buffer (0.1 M sodium phosphate, 0.1% Igepal Ca630, pH 8) at room temperature and 3 washes of 0.2x SSC, 0.1x SSC and 0.01x SSC at room temperature followed by centrifugation. Arrays were scanned using a laser scanner (ScanArray Express, Perkin Elmer) and analysed using ImaGene version 5.6. Cy5 cy3 ratios are calculating by taking the log2 of the signal mean of each spot minus the median background mean of all spots on the array. This is followed by a standard normalization for spot intensity and calculation of the ratios.
2010-06-05 | E-GEOD-2352 | biostudies-arrayexpress
Project description:Synechocystis 6803 cells was grown photoautotrophically at 32 °C buffered in BG-11 and bubbled with 3% CO2. A relatively mild Ci stress was applied by switching the aeration from 3% CO2 to air alone. After incubation under designated conditions, a 100-ml aliquot of culture was immediately combined with an equal volume of ice-cold mixture of phenol and ethanol (1:10, w/v) in an ice bath. The resultant cells were collected by centrifugation at 1000 x g for 10 min at 4 °C. Total RNA was isolated with RNeasy Midi Kit (Qiagen, Valencia, CA) and further treated with the DNA-free kit (Ambion, Austin, TX). Fluorescently labeled cDNA was produced via a two-step indirect procedure involving cDNA synthesis from 16 µg of total RNA in a reverse transcriptase reaction incorporating aminoallyl-modified deoxynucleotide, followed by the second step involving chemical coupling of fluorescent dye to the introduced amino moieties of the synthesized cDNA. Labeled cDNA were adjusted to 14.75 µl, and the remainder of the hybridization components containing 2.5 µl of 10 µg µl-1 salmon sperm DNA, 8.75 µl of 20x SSC, 0.25 µl of 10% SDS, and 8.75 µl of formamide were added. The mixture was then heated for 2 min at 99 °C and maintained at 42 °C until hybridization. Hybridizations were preformed in a static incubator at 42 °C for 12-16 h then washed by placing in a 250-ml solution of 2x SSC and 0.1% SDS at 42 °C for 5 min with gentle agitation provided by rotation of a magnetic stir bar. The slide was transferred quickly to a 250-ml solution of 0.1x SSC and 0.1% SDS, incubated for 10 min at room temperature with gentle agitation, and washed five additional times in 0.1x SSC for 1 min at room temperature. Hybridization signals from the microarray were quantified using GenePix Pro 4.1 (Axon Instruments, Union City, CA). The quality control procedures were conducted in the image analysis software, and then data were saved to Acuity 3.1 (Axon Instruments). Keywords: time-course
2004-08-24 | GSE1695 | GEO
Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other
2004-02-11 | GSE1031 | GEO
Project description:One day-old broilers (Ross/Ross, Longenecker's Hatchery, Elizabethtown, PA) were housed in Petersime starter brooder units and randomly assigned to 4 groups (5 birds/group). Carvacrol (Nature Chemicals, Hamburg, Germany) and cinnamaldehyde (Alys Technologies SA, Bussigny-près-Lausanne, Switzerland) were obtained from the compound obtained from synthesis. Crushed C. annuum fruits (Pushp Brand Spices, Munimji Foods and Spices Pvt. Ltd. Indore, India) were extracted with volatile solvents using the percolation method to obtain oleoresin. Chickens were fed for 7 days beginning from hatch with a standard diet alone (control) or with diets supplemented with carvacrol, cinnamaldehyde, or Capsicum oleoresin.<br> Following euthanization of the birds, intestines were cut longitudinally and washed three times with ice-cold Hanks? balanced salt solution (HBSS) containing 100 U/ml of penicillin and 100 mg/ml of streptomycin (Sigma, St. Louis, MO). The mucosal layer of entire intestine was carefully scraped using a surgical scalpel and intraepithelial lymphocytes (IELs) were isolated by Percoll density gradient centrifugation as previously described (Min et al. 2005). Total RNA was isolated from 5.0 x 107 cells using Trizol (Invitrogen, Carlsbad, CA) and purified using the RNeasy Mini RNA Purification Kit (Qiagen, Valencia, CA). Aminoallyl-labeled RNA from IELs was prepared using the Amino Allyl Message Amp II aRNA Amplification Kit (Ambion, Austin, TX). Two of 20 ?g aliquots of each aminoallyl-RNA sample were fluorescently labeled with AlexaFluor 555 or AlexaFluor 647 (Invitrogen) and labeled RNAs were column-purified using the RNA Amplification Kit (Ambion). RNA concentrations and labeling efficiencies were determined spectrophotometrically.<br> Hybridizations were performed using HybIt hybridization buffer (TeleChem, Sunnyvale, CA) in ArrayIt reaction cassettes at 50°C overnight as described (Kim et al., 2008). After hybridization, the slides were rinsed in 0.5 X SSC, 0.01% SDS at room temperature and washed once for 15 min in 0.2 X SSC, 0.2% SDS at 50°C, 3 times for 1 min in 0.2 X SSC at room temperature, and 3 times for 1 min in distilled water at room temperature. Each sample had a repeated hybridization using the alternate fluorescent dye between the treatment and control.
2009-06-02 | E-MEXP-2204 | biostudies-arrayexpress
Project description:Cutaneous melanomas are malignant radio and chemoresistant neoplasias presenting high morbidity and elevated mortality rates. Isolated Limb Perfusion (ILP) with Melphalan (Mel) is used in the treatment of non-resectable locally advanced melanomas of extremity sparing the limb from amputation in 40-50% of the cases. Adding the Tumor Necrosis Factor alpha (Tnfa) improves total complete response to 70-90%. The cellular and molecular mechanisms underlying the changes promoted by Mel and Tnfa are not completely understood. In this study we evaluated the impact of systemic Mel and Tnfa administration on tumor growth, analyzed the morphological changes promoted by each treatment, and searched for early molecular targets of Mel and Tnfa, alone or in combination, in a murine melanoma model. Morphological changes were analyzed by histological analysis, while microarray gene expression followed by quantitative RT-PCR were used to search for early molecular targets. We found that Mel systemic administration accounted for the impairment of tumor-growth (p<0.001) and improvement of survival. Tnfa co-administration augmented necrosis (p<0.024) and decreased mitotic rates (p=0.001). We identified a set of 118 potential molecular markers that might be correlated with the observed biologic response to treatment with Melphalan and Tnfa and which could represent potential therapeutic targets in melanoma. We used amplified RNA (aRNA) from 18 tumor samples (Control – 5 samples; Mel – 4 samples; Tnfa – 4 samples; and Mel+Tnfa – 5 samples). For replica hybridization with dye swap, aRNA from the tumors and from a pool composed of equal amounts of RNA extracted from the K1735M2, M2R, B16F10, and B16F1 melanoma cells were labeled with Alexa555 or Alexa647. Labeled aRNA samples were hybridized against a glass platform containing 16,128 immobilized sense oligonucleotides (Fox Chase Cancer Center, USA) corresponding to murine genes. Slides were pre-hybridized at 42ºC for approximately 6 hours in a solution containing Denhardt’s 5X (Ficoll 400 0.05g/mL, poli-vynil-pyrrolidone 0.05g/mL, bovine serum albumin 0.05g/mL), SSC (sodium saline citrate) 5X, 0.2% SDS (Sigma), and 1% BSA. For hybridization, we used a mix of 3.5µg of each labeled sample aRNA and reference aRNA. The hybridization solution contained Denhardt’s 5X, formamide 25% (Sigma), SSC 5X, SDS 0.1%, salmon sperm DNA 0.1mg/mL (GE Healthcare), Poly-A 0.1mg/mL (GE Healthcare), and Cot-1 0.1mg/mL (GE Healthcare), to a final volume of 100µL. Hybridizations were carried out on a Gene Tac hybridization station (Genomic Solutions) at 42ºC for about 16h. The slides were removed from the hybridization cassettes directly to a recipient containing a washing solution (SSC 2X, SDS 0.1%) at 42ºC, put in a slide rack, transferred to another recipient containing fresh solution, and washed under constant agitation at 42ºC for 5 min. After that, they were washed twice for 5min in a second wash solution (SSC 0.1X, SDS 0.1%) at room temperature and rinsed five times for 1min at room temperature with a SSC 0.1X solution. The slides were dried upside down in a centrifuge at 1500rpm for 5min. The slides were scanned with a confocal laser scanner (ScanArrayTM Express, Perkin-Elmer Life Sciences, USA), and the spot intensities processed with the help of the ScanArray Express program (Packard BioScience), with a 10µm resolution and a PMT of 60% for Alexa 555 and of 70% for Alexa 647. Each slide generated a data set for each channel corresponding to the dyes Alexa 555 and Alexa 647. The slides were scanned with a confocal laser scanner (ScanArrayTM Express, Perkin-Elmer Life Sciences, USA) with a 10µm resolution and a PMT of 60% for Alexa 555 and of 70% for Alexa 647, and data were extracted with ScanArray Express software (Packard BioScience) using the histogram method. Each slide generated a data set for each channel corresponding to the dyes Alexa 555 and Alexa 647. The Locally Weighted Scatter-plot Smoothing method (LOWESS), adjusted for linear and non-linear systematic variations, both of the intensity dependent type, mainly for low intensity spots, was employed for data normalization.
2011-12-31 | E-GEOD-21559 | biostudies-arrayexpress
Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon macrophages. Infected and control (24 h incubation) macrophage mRNA samples were subjected to two rounds of amplification using the MessageAmp aRNA kit (Ambion) and manufacturer's instructions. Approximately 400 ng of macrophage mRNA was used as template in the first round of amplification, yielding 8.1 ug of infected macrophage aRNA and 7.8 ug of control macrophage aRNA. Approximately 1 ug of first-round macrophage aRNA was used as template in the second round of amplification, yielding 81.2 ug of infected macrophage aRNA and 83.3 ug of control macrophage aRNA. Infected and control macrophage aRNA samples were visualized on agarose gels to check quality and quantity. Except for the amounts of aRNA and reverse transcription (RT) primer used, macrophage target labeling reactions and microarray hybridizations were identical. Macrophage target syntheses used 5 ug of second-round aRNA and 5 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5'T18[Q-N]3'), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 72 Cy3, PMT 63 or 64 Cy5 for macrophage study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other
2004-02-11 | GSE1012 | GEO
Project description:We have performed the hybridisation in triplicate using total RNA samples pooled from three independent batches of primary cortical neuron culture derived from E14.5 CD1 mouse brains on one day in vitro (1DIV). We stimulated the cells with 50 ng/ml FGF2 in the presence of 10 μg/ml heparin for 4 hours, and the expression of genes were compared to those in the absence of FGF2. The data were obtained from the three experiments with the best possible conditions and strict measures were applied for the purpose of quality control. Seventeen features representing housekeeping genes in the array (GAPDH, HPRT and S16, S9, and S8 ribosomal proteins) gave average value of Ratio of Medians (ROM), 1.20 ± 0.28, which was confirmed to be acceptable. Total RNA was extracted from cells in the growing phase by using RNA Clean (Hybaid) according to the manufacturer’s instruction and further purified with RNeasy mini kit (Qiagen). Direct labelling of 20 µg of total RNA primed with Oligo(dT) was performed in the presence of Cy-5 and Cy-3-dUTP (Amersham Pharmacia Biotech) using SuperScript II (Invitrogen). The Cy5- and Cy3-labelled cDNA samples were then mixed and purified with GFX DNA purification kit (Amersham Pharmacia Biotech). The samples were added with 1.2 µg of salmon sperm DNA and 5 µg of Poly(dA) and dried in SpeedVac at 60°C. The high-density glass microarray chips were prepared at the EMBL core facility by spotting the PCR products prepared from the NIA 15k cDNA mouse clone set. Array slides were pre-hybridized in 6x SSC, 0.5% SDS, 1% BSA at 42°C for 1 hour and incubated in a boiled water for 2 min shortly prior to hybridisation. The cDNA samples were resuspended in 15 µl of hybridisation buffer (50% formamide, 6x SSC, 0.5% SDS and 5% Denhardt’s) and denatured by incubating at 95°C for 2 min. Hybridisation was at 42oC for 16 hours. The slides were washed for 10 min each with 2 x SSC, then with 0.5 x SSC, 0.1%SDS, followed by 0.1 x SSC, 0.1%SDS, at room temperature. Scanning was performed with GenePix 4000B (Axon). Quality control was performed, firstly by eye, to confirm scanner alignment and absence of significant bubbles and scratches. Scatter plots were further used to eliminate the unacceptable hybridisation data. The multiple spike-in controls RNA template were added to the sample upon direct labelling, and the successful labelling and hybridisation was confirmed in each hybridisations. GenePix Pro grogram calculates the Normalisation Factor of each hybridisation, based on the premise that the arithmetic mean of the ratios from every feature on the given array should be equal to 1. Normalisation was therefore performed by multiplying the Factor to Ratio of Medians (ROM) in each gene. The program also identifies features that did not give good alignment to the expected spotted area as “flagged” spots, indicative of the impaired-quality hybridisation of the specific genes. Keywords = NIA mouse 15k Keywords = cortical neuron Keywords = FGF Keywords: other
2004-12-15 | GSE2066 | GEO
Project description:Examination of molecular basis of malaria resistance. Spleens from malaria resistant AcB55 and AcB61strains compared with A/J mice. Animals were sacrificed and spleens were rapidly frozen in liquid nitrogen, and subsequently stored at 80oC. Tissues were mechanically homogenized with a polytron and total cellular RNA was extracted using a commercially available reagent (TMTRIzol; Invitrogen). The poly-(A)+ fraction of the RNA was isolated by chromatography with oligo d(T) cellulose beads. For cDNA labeling, either 25 mg of total RNA or 2.5 mg of poly-(A) RNA was converted into cDNA using reverse transcriptase (RT; Super Script II, Invitrogen) and alternatively Cy5 or Cy3-labeled dCTP (1mM, Perkin Elmer-Cetus/NEN, Boston) in a reaction mixture containing 1.5 mL oligo (dT) (100pmol/ mL), 3 mL dNTP-dCTP (6.67mM each), 1 mL dCTP (2mM), 4 mL DTT (100mM), 8 mL 5 X RT Buffer (Invitrogen, California). The reactions were carried out at 42¼C for 3 hrs, after which the RNA was degraded by the addition of 0.5 mL RNase A (1 mg/mL) and 1.5 mL RNaseH 5 units/mL). Labeled cDNA was separated from unincorporated nucleotides (Qiagen column) and further concentrated by evaporation under vacuum. The arrays were pre-hybridized for 1-2hrs with DIGEasy hybridization buffer (Roche) containing 10ug/ml denatured salmon sperm DNA, and 10ug/ml yeast tRNA. The Cy5 and Cy3 labelled cDNAs were combined and hybridized in the same medium and incubated with the arrays for 16-18hrs at 37Co. Finally, the arrays were washed 3 x 10 mins in 0.1 x SSC (20 X SSC is 3M sodium chloride, 0.3 M sodium citrate, pH 7.0), 0.1%SDS at 50Co, and 4 x 3min in 0.1 x SSC at room temperature, and dried by centrifugation. Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray fluorescent scanner and image intensity data were extracted and analyzed with QuantArray 3.0 analysis software. Quantification data was imported in GeneSpring (Silicon Genetics). The import format was modified to maintain information relevant to user-contributed flagging as well as QC parameter furnished by the array manufacturer. Dye swap and Intensity-based normalization (Lowess) were performed using the default settings. Keywords: other
2003-10-06 | GSE709 | GEO
Project description:Ten male 4get mice were infected with N. brasiliensis and single-cell suspensions of total lung tissue were prepared 9 days after infection. Cells were stained for CD3, CD19, c-kit and CCR3 and sorted for CD3-CD19-c-kit-GFP+CCR3+SSChi (eosinophils) and CD3-CD19-c-kit- GFP+CCR3-SSClo (basophils) using a MoFlo high-speed cell sorter (Cytomation, Fort Collins, CO). Total RNA was isolated from 2 x 106 eosinophils and 1.75 x 105 basophils using the Total RNA Isolation Kit (Fluka, Buchs, Switzerland) and amplified by two rounds of in vitro transcription using the Amino Allyl MessageAmpTM aRNA kit (Ambion, Austin, TX). Universal Mouse Reference RNA (#740100; Stratagene, La Jolla, CA) was amplified in the same way. Aminoallyl-UTP was incorporated during the second round of amplification and 5 µg of amplified RNA was coupled to Cy3 and Cy5 fluorescent dyes (CyScribeTM dye labeling kit, Amersham Biosciences, Peapack, NJ). Probes were hybridized to spotted glass oligonucleotide (70-mer) arrays which cover just over 16,400 unique genes (Mouse Genome Set Version 2.0, Qiagen, Germany). The arrays were prehybridized in 1% BSA (Invitrogen), 5 x SSC, 0.1% SDS for 2 hr at 42°C, washed in water and hybridized with the Cy3/Cy5-labeled probes in 2.8 x SSC, 0.2% SDS, 0.6 mg/ml Cot-1 DNA (Invitrogen), and 0.8 mg/ml yeast tRNA, at 63°C for 45 hr. Slides were washed sequentially in 0.03%SDS/1 x SSC, 0.2 x SSC and 0.05 x SSC, dried and scanned on an Axon 4000B scanner using Genepix 3.0 software (Axon Instruments, Inc.). Data were normalized using the R package Bioconductor software and ?lowess? normalization on the pixel medians without background subtraction (Ihaka and Gentleman, 1996). Keywords = Eosinophils Keywords = Basophils Keywords = Nippostrongylus Keywords = Lung Keywords = Type 2 immunity Keywords: other
2004-02-21 | GSE984 | GEO
Project description:Purpose: Utilized Next-generation sequencing (NGS) to profile transcriptional differences between two populations, carriers (CC) of rs10846744 SNP associated with cardiovascular disease (CVD) and non-carriers (GG) who are disease free. Methods: Total RNA was isolated from three subjects homozygous for the rs10846744 reference (GG) allele and three subjects homozygous for the rs10846744 risk (CC) allele and then subjected to full transcriptome sequencing using the Perkin Elmer next gen sequencing platform (Perkin Elmer, Branford, CT). Bioinformatics was performed using Perkin Elmer GeneSifter software program. The data was adjusted by selecting total map reads, quality reads >20, log transformation, and using Benjamini Hochberg to correct for multiple testing. RNA targets of interest were validated by qRT–PCR using TaqMan assays and western blotting using standard methodologies. Results: Using Perkin Elmer's Genesifter Analysis Edition Software, we mapped about 100 million sequence reads per sample to the human genome (build 37.2), normalized the raw read count by total mapped million reads and identified 937 upregulated and 587 downregulated transcripts in the EBV (Epstein Barr Virus)-transformed B lymphocyte cells isolated from 3 carriers of the risk (CC) allele and 3 non-carriers of the (GG) reference allele with BWA workflow. RNA-seq data confirmed differential expression of LAG3 and this was validated with qRT–PCR. Conclusions: Our study represents the first detailed analysis of differential LAG3 expression, contributing to CVD, with biologic replicates, generated by RNA-seq technology.
2016-10-20 | GSE87891 | GEO