title: Temperature validation experiment for custom array production

status: Public on Dec 31 2009

city: Antwerpen

first: Dries

last: Knapen

address: Groenenborgerlaan 171

institute: University of Antwerp

summary: Microarrays have evolved from low-density cDNA or oligonucleotide arrays to high-density platforms, for several study species even covering the complete transcriptome. At the same time, transcriptomics experiments have become more complex and multifactorial in nature, requiring many microarrays to assess multiple biologically relevant hypotheses. Scientists using this technology are therefore painfully aware of the high financial cost of a typical microarray experiment. Unfortunately, this often leads to either a suboptimal experimental design in an effort to reduce the cost by using fewer microarrays, or to abandoning microarray technology altogether. In this study, we argue that for many studies high-density full genome microarrays are in fact technical overkill. By selectively reducing full genome probe sets to a lower number of probes, it is possible to significantly reduce the total cost of a microarray experiment. The study consists of four microarray analyses: a cadmium probe selection experiment, a temperature probe selection experiment, a cadmium validation experiment and a cadmium validation experiment.

overall_design: Samples were hybridized in a simple loop design, in which every sample was labelled once with the red dye and once with the green dye. The hybridization design for the temperature validation experiment was (T26D4R2 > T34D4R1 > T26D4R3 > T34D4R2 > T26D4R2). Arrows (>) indicate individual microarrays; heads represent the green dye and tails represent the red dye. Sample names are of the form TaDbRc (temperature a in °C, day b, biological replicate c).

first: Lucia

last: Vergauwen

first: Ronny

last: Blust


title: Temperature probe selection experiment for custom array production

department: Biology

country: Belgium

overall_design: Samples were hybridized in a simple loop design, in which every sample was labelled once with the red dye and once with the green dye. The hybridization design for the temperature probe selection experiment was (T26D28R1 > T34D28R1 > T18D28R1 > T26D4R1 > T34D28R2 > T26D28R2 > T18D28R2 > T26D28R1 + T26D28R2 > T34D28R1). Arrows (>) indicate individual microarrays; heads represent the green dye and tails represent the red dye. Sample names are of the form TaDbRc (temperature a in °C, day b, biological replicate c)


title: Foxf1 enchances pulmonary inflammation and mastocytosis

status: Public on Sep 04 2008

city: Chicago

first: Tanya

last: Kalin

state: IL

address: 5841 S. Maryland Ave.

department: Medicine

country: USA

institute: University of Chicago

summary:
The Forkhead Box f1 (Foxf1) transcriptional factor (previously known as HFH-8 or Freac-1) is expressed in endothelial and smooth muscle cells in the embryonic and adult lung. To assess effects of Foxf1 during lung injury, we used CCl4 injury model.  Foxf1+/- mice developed severe airway obstruction and bronchial edema, associated with increased numbers of pulmonary mast cells and increased mast cell degranulation following injury.  Pulmonary inflammation in Foxf1+/- mice was associated with diminished expression of Foxf1, increased mast cell tryptase and increased expression of CXCL12, the latter being essential for mast cell migration and chemotaxis.  Foxf1 haploinsufficiency caused pulmonary mastocytosis and enhanced pulmonary inflammation following chemically-induced lung injury, indicating an important role for Foxf1 in the pathogenesis of pulmonary inflammatory responses.
Keywords: Influence of genetic modification on the pulmonary inflamation

overall_design: Foxf1+/- mice in which the Foxf1 allele was disrupted by an in-frame insertion of a nuclear localizing beta-galactosidase (B-Gal) gene were bred for ten generations into the Black Swiss mouse genetic background. Carbon tetrachloride (CCl4; Sigma, St Louis, MO) was dissolved in mineral oil at a 1:20 ratio v/v and a single intraperitoneal  (i.p.) injection of CCl4 (0.5 microliter of CCl4/ 1g of body weight) was administered to male Foxf1+/- mice or their wild type (WT) littermates as described.

middle: V

first: Lucille

last: Meliton

first: Angelo

first: Xiangdong

last: Zhu

first: Jeffrey

middle: A

last: Whitsett

first: Vladimir

last: Kalinichenko


title: Cadmium validation experiment for custom array production

overall_design: Samples were hybridized in a simple loop design, in which every sample was labelled once with the red dye and once with the green dye. The hybridization design for the cadmium validation experiment was (C0D4R2 > C5D4R1 > C0D4R3 > C5D4R2 > C0D4R2). Arrows (>) indicate individual microarrays; heads represent the green dye and tails represent the red dye. Sample names are of the form CxDyRz (cadmium concentration x in µM, day y, biological replicate z).


title: Hornfly 1st instar larvae vs pooled adult + egg

status: Public on Jun 19 2008

city: Lubbock

first: Scot

last: Dowd

state: TX

address: 4321 Marsha Sharp Fwy

department: MBRI

institute: MBRI

summary:
Background: The horn fly, Haematobia irritans (L.), is an obligate blood-feeding parasite of cattle and control of this pest is a continuing problem in the United States and other parts of the world. Worldwide annual economic losses attributable to this pest surpass $1 billion. The fly is becoming resistant to pesticides and new control technologies are needed by cattle producers.

Results: Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene. To identify candidate genes to supply this gene promoter, microarrays were designed from a recently developed horn fly EST database and probed to identify female-specific and larval-specific differential gene expression. Analysis of dyeswap experiments found 432 and 417 transcripts which were over- and under-expressed in adult female flies, respectively, compared to adult male flies. Additionally, 419 and 871 transcripts were over- and under-expressed in first instar larvae compared to adult flies. Three transcripts were identified which were over-expressed in adult females flies compared to adult males and which also were over-expressed in the first instar larval lifestage compared to adult flies.

Conclusion: We have identified 3 strong candidates for further evaluation as a gene promoter source for the development of a female-specific conditional lethality system in the horn fly. One of these candidates, the putative nanos orthologue, has a high female-to-male expression ratio, has a moderate expression level in first instar larvae, and has been well characterized in D. melanogaster. Further investigations leading from this microarray analysis will include transformation of the horn fly and evaluation of the female conditional lethal system components for applicability to the specific biological parameters of natural populations of the horn fly.
Keywords: evaluation of 1st instar larvae genes vs pooled adult + egg genes

overall_design: In short, RNA from female horn flies was labeled and hybridized against labeled RNA from male horn flies in a dyeswap design. Similarly, RNA from first instar larvae was labeled and hybridized against labeled a 50:50 mix of RNA from adult male and female flies. All labeling, hybridization and washing procedures were performed according to respective manufacturer protocols. For each sample, 10 ug of total RNA and 50-fold dilution of Agilent two color spike-in control RNA kit (Agilent Technologies Inc.) were labeled with either CyDye3-dCTP or CyDye5-dCTP (Amersham Biosciences, Piscataway, NJ) using the LabelStar kit (Qiagen Inc., Valencia, CA) and oligo-dT and Random nonamers (Sigma-Aldrich Inc., St. Louis, MO). Labeled cDNA was hybridized to the Agilent microarrays using the Gene Expression Hybridization kit (Agilent Technologies Inc.) following manufacturer’s protocols. Arrays were washed with Gene Expression Wash Buffer kit (Agilent Technologies Inc.) A total of 8 arrays, including a dye swap for each RNA replicate (4 biological replicates), were analyzed to obtain genes that were consistently regulated while limiting false discover rate (FDR) below 2.5% [26].

middle: E

first: Felix

last: Guerrero


title: Mesenchymal stem cells from myelodysplastic syndromes are functionally and genomically abnormal

status: Public on Aug 01 2008

city: Salamanca

first: Olga

last: Lopez

address: P/ San Vicente 58-182

department: Hematology

country: Spain

institute: Hospital Universitario de Salamanca

type: Genome variation profiling by genome tiling array

summary:
Myelodysplastic syndromes (MDS) are a group of clonal disorders of hematopoietic stem cells. Mesenchymal stem cells (MSC) are the progenitors of the Bone Marrow (BM) stroma and have been involved in the physiopathology of MDS. The presence of cytogenetic aberrations on MSC from MDS patients is controversial. The aim of the study is to characterize BM derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridisation (FISH) analysis and genetic changes by array based comparative genomic hybridization (array-CGH). 32 cases of untreated MDS were included in the study. MSC from MDS achieved confluence at a slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was also lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic, and gains were more frequent than looses. The results of array-CGH were confirmed by FISH. When an unsupervised hierarchical cluster analysis was performed two clusters were identified: one of them included the 5q- syndrome patients, while the other incorporated the rest of the MDS patients. Our results shows, for the first time, that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to particular MDS subtypes.
Keywords: Genomic comparison between mesenchymal cells

overall_design:
32 BM cells from ?de novo? MDS, corresponding to 29 patients, were included in the study. Male/female ratio was 14/15 and median age was 70 years (range 34-89). MDS diagnosis was established by peripheral blood counts and morphology, BM morphology and cytogenetic analyses and classified according to the WHO proposals. 12 Patients? characteristics are summarized in table 1. Three patients were included twice in the study: at the diagnosis of one RA (case 2) and two Unclassifiable MDS (cases 29,30), and at the progression into RAEB-2 (cases 16, 20 and 18). None of them had received treatment during this period of time, other than supportive therapy. Normal BM samples were obtained from 15 healthy donors, 8 males and 7 females, with a median age of 57 (range 37-88). Written informed consent was obtained from all patients and donors according to ethical guidelines of our institution.
Genomic-wide analysis of DNA copy number changes of MSC from 13 cases was performed using array-CGH. Slides containing 3032 BACs were produced in ?Centro de Investigación del Cáncer? (Salamanca, Spain). The particular BAC and P-1 derived artificial chromosome set used to produce this array is distributed to academic institutions by the Welcome Trust Sanger Institute (Cambridge, United Kingdom) and contains targets spaced at about 1 Mb density over full genome, a set of subtelomeric sequences for each chromosome arm, and a few hundred probes selected for their involvement in oncogenesis. The clone content is available in the ?Cytoview? windows of the Sanger Institute mapping database site, Ensembl (http://www.ensembl.org/). According to this database, clones were ordered along the chromosomes. These clones were isolated from their bacterial cultures with the relevant antibiotics and the DNA was extracted with standard protocol Welcome Trust Sanger Institute (Cambridge, UK). 10 ng of DNA (BAC/PAC) was used as a template for three DOP-PC. 22;23 These products were ethanol precipitated and dissolved in distilled water. A minimum of three replicates per clone were printed by Microgrid II (Biorobotics) on each slide (Ultragaps Coated Slides, Corning) using the aqueous dimethylsulfoxide buffer as spotting solution. Briefly, for labelling reactions, 1 ?g of nonamplified genomic DNA, test and reference were digested separately with DpnII restriction enzyme (New England Biolabs, Beverly, MA). For microarray hybridization, the digested DNAs were separately labelled using random primers (Bioprimer labelling kit, Invitrogen) and cyanine dye-3-dCTP and cyanine dye-5-dCTP fluorescent dye to paired hybridisation samples (Amersham Biosciences). The incorporated of the label nucleotide was quantified using the Nanodrop spectrophotometer. Labelled test and reference DNAs were mixed equitably, co-precipitated in presence of Cot-1 human DNA (Roche, Indianapolis, IN) with ethanol, washed, and resuspended in hybridization solution (50% Formamide, 10% Dextran sulfate, 2X standard saline citrate, 10 mM Tris pH 7.6, 2.7% sodium dodecyl sulfate and 10 ?g/ ?l of yeast tRNA). DNA mixtures were hybridized to the arrays in the TECAN HS4800 Pro according to the manufacturer?s recommended protocol. Images and signal intensities were acquired using GenePix4000B (Axon Instruments, Burlingame, CA) dual laser scanner in combination with GenePixPro4.0 (Axon Instruments) imaging software.
Image and Data analysis.
Spot intensities measured by the GenePix Pro 4.1 software (Axon Instruments, Wetsbur BV, Leuden, The Netherland). Pixel intensities for each feature were integrated and median values were determined, and the local background was calculated. For each spot the intensities were corrected by subtracting the local background for both wavelengths, and only spots with signal intensities al least threefold above background signal intensities were included in the analysis. The median of the ratios of all spots was calculated and used to normalize all data points. From each feature, the log2 value of the average of the normalization ratios of the spots was calculated and used to display the data. In addition, fluorescence ratios were normalized for each microarray using the print-tip loess method and the background subtraction with the Diagnosis and Normalization Arrays Data tool. 24 The cut-off level was determined for each individual experiment after avering the ratios from the two color-swith hybridizations and subsequent for normalization, and calculate the mean and Standard derivations to define the cut-off level as means plus/minus two times the standard derivations. The clones that showed possible changes were submitted to the Database of Genomics Variants (http://projects.tcag.ca/variation/) and chromosomal Imbalances and Phenotype in Humans using Ensembl Resources (DECIPCHER: http: www sanger. ac.uk /PostGenomics/decipher/.
Clustering
Unsupervised clustering
We converted the relative ratio value for each BAC clones in each sample to score of 1 (gain/amplified), 0 (no change), or -1 (loss) based on the 2 SD standards described above and analyzed with Cluster and TreeView) 25 (Cluster and TreeView software) based on the average linkage method with the centered correlation metric was used (or Pearson Uncentered).
Supervised
Sam analysis was used http://www-stat.stanford.edu/ tibs/SAM) 26 to identify BACs that segregate subgroups on MSC. BACs with an SD of zero across the data set before analysis were removed.

last: Lopez-Villar

first: Juan

middle: L

last: Garcia

first: Fermin

middle: M

last: Sanchez-Guijo

first: Cristina

last: Robledo

first: Eva

last: Villaron

first: Natalia

last: Lopez-Holgado

first: Maria

last: Diez-Campelo

first: Victoria

last: Barbado

first: Jose

last: Perez-Simon

first: Jesus

last: Hernandez-Rivas

middle: F

last: San Miguel

first: Consuelo

last: Cañizo


title: ChIP-chip Designs to Interrogate the Xenopus Embryo Genome for Transcription Factor Binding and Epigenetic Regulation

status: Public on Dec 17 2009

city: Nijmegen

first: Gert Jan

last: Veenstra

address: Geert Grooteplein 28

department: Molecular Developmental Biology

country: Netherlands

institute: Radboud University

type: Genome binding/occupancy profiling by genome tiling array

summary: Background: Chromatin immunoprecipitation combined with genome tile path microarrays or deep sequencing can be used to study genome-wide epigenetic profiles and the transcription factor binding repertoire. Although well studied in a variety of cell lines, these genome-wide profiles have so far been little explored in vertebrate embryos.   Principal findings: Here we report on two genome tile path ChIP-chip designs for interrogating the Xenopus tropicalis genome. In particular, a whole-genome microarray design was used to identify active promoters by close proximity to histone H3 lysine 4 trimethylation. A second microarray design features these experimentally derived promoter regions in addition to currently annotated 5’ ends of genes.   Conclusions: A whole-genome and a dedicated promoter microarray design was developed which can be used to study epigenetic phenomena and transcription factor binding in developing Xenopus embryos.

overall_design: H3K4me3 and TBP ChIP-chip on Xenopus tropicalis tiling arrays

first: Robert

middle: C

last: Akkers

first: Simon

middle: J

last: van Heeringen

first: J

middle: R

last: Manak

first: Roland

middle: D

last: Green

first: Hendrik

middle: G

last: Stunnenberg

first: Gert


title: BXD Mouse Liver Gene Expression

status: Public on Aug 06 2009

city: Chapel Hill

first: Daniel

last: Gatti

state: NC

address: 135 Dauer Dr.

department: Environmental Sciences & Engineering

institute: University of Noth Carolina

summary:
The liver is the primary site for the metabolism of nutrients, drugs, and chemical agents. Although metabolic pathways are complex and tightly regulated, genetic variation among individuals, reflected in variations in gene expression levels, introduces complexity into research on liver disease. This study dissected genetic networks that control liver gene expression through the combination of large-scale quantitative mRNA expression analysis with genetic mapping in a reference population of BXD recombinant inbred mouse strains for which extensive single-nucleotide polymorphism, haplotype, and phenotypic data are publicly available. We profiled gene expression in livers of naive mice of both sexes from
C57BL/6J, DBA/2J, B6D2F1, and 41 BXD strains using Agilent oligonucleotide microarrays. These data were used to map quantitative trait loci (QTLs) responsible for variations in the expression of about 19,000 transcripts. We identified polymorphic local and distant QTLs, including several loci that control the expression of large numbers of genes in liver, by comparing the physical transcript position with the location of the controlling QTL. CONCLUSION: The data are available through a public web-based resource (www.genenetwork.org) that allows custom data mining, identification of coregulated transcripts and correlated phenotypes, cross-tissue, and cross-species comparisons, as well as testing of a broad array of hypotheses.

overall_design: Basal gene expression in the livers of 41 BXD strains, the parentals and the F1 cross, consisting of C57BL/6J, DBA/2J, B6D2F1, BXD1, BXD2, BXD5, BXD6, BXD8, BXD9, BXD11, BXD11TY, BXD12, BXD13, BXD14, BXD15, BXD16, BXD19, BXD21, BXD23, BXD24, BXD28, BXD29, BXD31, BXD32, BXD33, BXD34, BXD36, BXD38, BXD39, BXD40, BXD42, BXD43, BXD44, BXD45, BXD48, BXD51, BXD60, BXD62, BXD69, BXD73, BXD77, BXD85, BXD86, BXD92 pooled from 2-3 mice of the same sex and strain. The arrays were run in 8 batches, balanced by sex and strain with intra- and inter-batch replicates. Interbatch normalization was carried out using a nested analysis of variance mixed model, with samples within each batch crossed with sex and strain.

first: Ivan

last: Rusyn


title: Clinical validation of a customized multiple signature microarray for breast cancer

status: Public on Jul 23 2008

city: Singapore

first: Kun

last: Yu

department: Cellular & Molecular Research

country: Singapore

institute: National Cancer Centre

summary: PURPOSE: Current histopathologic systems for classifying breast tumors require evaluation of multiple variables and are often associated with significant interobserver variability. Recent studies suggest that gene expression profiles may represent a promising alternative for clinical cancer classification. Here, we investigated the use of a customized microarray as a potential tool for clinical practice. EXPERIMENTAL DESIGN: We fabricated custom 188-gene microarrays containing expression signatures for three breast cancer molecular subtypes [luminal/estrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2), and "basaloid"], the Nottingham prognostic index (NPI-ES), and low histologic grade (TuM1). The reliability of these multiple-signature arrays (MSA) was tested in a prospective cohort of 165 patients with primary breast cancer. RESULTS: The MSA-ER signature exhibited a high concordance of 90% with ER immunohistochemistry reported on diagnosis (P < 0.001). This remained unchanged at 89% (P < 0.001) when the immunohistochemistry was repeated using current laboratory standards. Expression of the HER2 signature showed a good correlation of 76% with HER2 fluorescence in situ hybridization (FISH; ratio > or =2.2; P < 0.001), which further improved to 89% when the ratio cutoff was raised to > or =5. A proportion of low-level FISH-amplified samples (ratio, 2.2-5) behaved comparably to FISH-negative samples by HER2 signature expression, HER2 quantitative reverse transcription-PCR, and HER2 immunohistochemistry. Luminal/ER+ tumors with high NPI-ES expression were associated with high NPI scores (P = 0.001), and luminal/ER+ TuM1-expressing tumors were significantly correlated with low histologic grade (P = 0.002) and improved survival outcome in an interim analysis (hazard ratio, 0.2; P = 0.019). CONCLUSION: The consistency of the MSA platform in an independent patient population suggests that custom microarrays could potentially function as an adjunct to standard immunohistochemistry and FISH in clinical practice.

overall_design: In the operating theater, morphologically visible tumor were removed by surgery and examined by a surgical pathologist to confirm the presence of cancer cells by cryosections. The tissue samples were divided into discrete aliquots, flash frozen and subsequently stored in liquid nitrogen

first: Patrick

last: Tan

first: Angie


title: The effects of estrogen and testosterone on gene expression in the rat mesenteric arteries

status: Public on Sep 30 2007

city: Vermillion

first: Kathleen

last: Eyster

state: SD

address: 414 E. Clark St.

department: Basic Biomedical Sciences

institute: University of South Dakota

summary:
Goal of the experiment:  To examine the effects of estrogen and testosterone on gene expression in the rat mesenteric arteries.

Brief description of the experiment:  A dramatic difference exists in the timing of development of cardiovascular disease in men vs. women.  The primary candidates underlying the cause of gender differences in cardiovascular disease are the sex steroids, estrogen and testosterone.  The effect of estrogen on the cardiovascular system has been expected to be protective but this concept has become controversial.  The effect of testosterone on the cardiovascular system is considered to be deleterious.  In spite of these concepts there is little data on the direct effects of estrogen and testosterone on gene expression in the vasculature.  Since estrogen and testosterone exert many of their effects through genomic mechanisms, the DNA microarray is an excellent tool for assessing their effects in the vasculature.  In this study, ovariectomized Sprague Dawley rats were treated for 4 days with vehicle (sesame oil), estradiol benzoate (0.15 mg/kg/day), or testosterone (1 mg/kg/day).  The mesenteric arteries were obtained, total RNA was extracted, and CodeLink Uniset Rat I DNA microarrays (GE) were used to identify differential gene expression.  Seven genes were identified as differentially expressed from the DNA microarray data and confirmed by real time RT-PCR.  The expression of D site albumin promoter binding protein and fatty acid synthase were increased in response to both estrogen and testosterone.  3alpha-hydroxysteroid dehydrogenase, interleukin 4 receptor, JunB and c-Fos expression were increased by estrogen but not by testosterone.  The aryl hydrocarbon nuclear translocator-like was reduced by testosterone.  These data identify genes not previously known to be responsive to estrogen and testosterone in the vasculature.

Keywords:  hormone treatment, vasculature, gender differences

Experimental factors:  hormone treatment
Keywords: hormone treatment

overall_design:
Experimental design:  3 treatments were given (vehicle (control), estradiol benzoate, and testosterone) and 4 animals received each treatment.  The mesenteric arteries were obtained from each of the 12 animals treated after euthanasia, total RNA was extracted from each mesenteric artery preparation, and cRNA was synthesized from each total RNA preparation (see below).  Samples from vehicle-treated animals are designated C for control:  C3, C4, C5, and C8.  Samples from estradiol benzoate-treated animals are designated E:  E2, E3, E5, and E8.  Samples from testosterone-treated animals are designated T:  T2, T4, T6, and T8.  Samples labelled AA and AB (i.e., C3AA, C3AB) are technical replicates derived from the same cRNA.

Quality control steps:  The cRNA that was synthesized from each mesenteric artery sample was used for hybridization to duplicate CodeLink (Amersham/GE) microarrays.  Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary.  Bacterial control spikes were used as per manufacturer's instructions.

Samples used, extract preparation and labelling:
The origin of each biological sample:  All samples were obtained from ovariectomized 5.5-week old female Sprague Dawley rats (Rattus norvegicus).

Manipulations of biological samples and protocols used:  The rats were ovariectomized at 3 weeks of age.  Hormone treatments were begun after a two week recovery period.  The rats were injected intraperitoneally with vehicle (sesame oil), estradiol benzoate (0.15 mg/kg/day), or testosterone (1 mg/kg/day) for 4 days.  The animals were euthanized after the treatment on the fourth day.  The mesenteric vasculature was dissected free from the gastrointestinal tract and placed in RNAlater (Ambion).  The mesenteric arteries were dissected free from the surrounding fat, mesenteric veins, and connective tissues while immersed in RNAlater (Rodrigo et al., J Pharm Tox Meth 47(2):87-92, 2002).

Experimental factor:  hormone treatment.

Technical protocols:  The mesenteric arteries were homogenized in TRI reagent (MRC) using a Polytron (Brinkmann).  The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen).  The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen).  The purity and quantity was evaluated by spectrophotometry and agarose gel electrophoresis.
Labelled cRNA was prepared using the manufacturer's Instruction Protocol (www1.amershambiosciences.com, CodeLink Gene Expression System: Manual Labelled cRNA Target Preparation).  The source of reagents was the CodeLink Expression Assay Reagent Kit, Manual Prep, except where specified otherwise.  2 micrograms of total mesenteric artery RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C.  (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.)  The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C.  The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C.  The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator.  The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA.  The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNeasy columns (Qiagen).  The concentration of cRNA was determined by spectrophotometry.

Hybridization procedures and parameters:  10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min.  The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C.
The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature.  The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer)  The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each.  The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

first: Connie

last: Mark

first: Richard

last: Gayle

first: Douglas

middle: S

last: Martin


title: Priming defense genes and metabolites in hybrid poplar by the green leaf volatile cis-3-hexenyl acetate

status: Public on Jul 24 2008

city: Gainesville

first: Christopher

last: Dervinis

state: FL

address: 2033 Mowry Road, rm 320

department: SFRC

institute: University of Florida

summary:
• Herbivore-induced plant volatiles (HIPVs), in addition to attracting natural enemies of herbivores, can serve a signaling function within plants by acting as wound signals that induce or prime defenses.  However, particularly in woody plants, which compounds within HIPV blends are capable of acting as signaling molecules are largely unknown.
• Leaves of hybrid poplar (Populus deltoides x nigra) saplings were exposed in vivo to naturally wound-emitted concentrations of the green leaf volatile (GLV) cis-3-hexenyl acetate (z3HAC) and then subsequently fed upon by gypsy moth larvae (Lymantria dispar L.).  Volatiles were collected throughout the experiments, and leaf tissue was collected to measure phytohormone levels and expression of defense-related genes.
• Relative to controls, z3HAC-exposed leaves had higher levels of jasmonic acid and linolenic acid following gypsy moth feeding.  Further, z3HAC primed transcripts of phytohormone signaling (lipoxygenase 1) and direct defense (a Kunitz proteinase inhibitor) genes. These qRT-PCR results were supported by microarray analysis using the AspenDB 7K EST microarray containing ~5400 unique gene models.  Moreover, z3HAC also primed the release of herbivore-induced terpene volatiles.
• The widespread priming response suggests an adaptive benefit to detecting z3HAC as a wound signal. Thus, woody plants can detect and use z3HAC as a signaling cue to prime defenses before actually experiencing damage.  GLVs may therefore have important ecological functions in arboreal ecosystems.

overall_design:
Plants and Gypsy Moths
Hybrid poplar (Populus deltoides x nigra, clone “OGY”: Magnoliopsida; Malpighiales; Salicaceae; Aigeiros section of Populus) saplings were grown from cuttings in a walk-in growth chamber maintained at 25oC with a 16:8 photoperiod. Cuttings were planted in five-gallon pots in commercial potting soil (MetroMix 250, SunGro, Bellevue, WA, USA) and watered as necessary. Due to supplements in the soil, fertilization was not required and poplars grew to heights of ~1.5 m in 12-15 weeks.
Newly molted 4th instar L. dispar larvae were used for all experiments. Egg masses were obtained from APHIS (Otis Plant Protection Lab, Cape Cod, MA, USA) and reared through 3rd instar on artificial diet. Newly molted 4th instar larvae cleared their gut contents prior to molting.

Experimental Manipulations
All experiments were conducted from March-April 2007 in a single walk-in growth chamber maintained as described above. Poplars were equilibrated to the chamber for 24 hours prior to manipulations.  Two fully-expanded source leaves (leaf plastochron index LPI 8 and 9) were isolated per sapling using individual 15x15x1 cm Teflon/glass chambers (for photo see Appendix in Frost et al., 2007). These two leaves are non-orthostichous, which means that they do not readily share assimilates or wound signals (Davis et al., 1991). Thus, for the purposes of these experiments, treatment leaves did not interact with their respective control leaves. However, the leaves are of slightly different ages and, to control for this potential effect, we randomized which leaf would receive the z3HAC treatment. Flow into each chamber was set at 2.0 L/min, which was sufficient to avoid condensation within the chamber.
The experiments were designed to have two consecutive albeit separate treatments: exposure to z3HAC and subsequent application of L. dispar larvae. We used commercially available z3HAC (Sigma-Aldrich), and prepared concentrations to infuse into each septum such that it delivered ~40 ng z3HAC per application. After a pretreatment volatile collection (Day 1 and night starting Day 2), z3HAC-infused rubber septa were placed into the leaf chambers with each treatment leaf (morning of Day 2). Control leaves each received a septum infused with just the dichloromethane solvent.  Septa were replaced twice, once before the afternoon collection on Day 2, and before the morning collection on Day 3. All septa were removed during the night of Day 3, since most volatile production stops at night in this hybrid (Fig 5a). On the morning of Day 4, two newly molted 3rd or 4th instar gypsy moth larvae were added to each leaf, regardless of whether the leaf received the treatment or control septa on Days 2 and 3. The herbivores remained on the leaves for ~24 hours.
On the day of collection, each leaf was excised at the petiole, photographed, and then placed in a labeled coin envelope and submerged directly into liquid N2. Each photograph also contained a standard of known area, from which leaf area and % damage calculations were determined (Sigma Scan 500, Systat Software Inc.).  Larvae consumed similar amounts on the treatment and control leaves (24 hours: z3HAC-treated leaves: 4.7 ± 1.8 cm2; controls: 4.3 ± 1.7 cm2).  The snap-frozen leaves were stored at -80oC until grinding. Each leaf was ground in an individual mortar and pestle under liquid N2. Ground tissue was weighed into vials for RNA extraction and JA analysis (see below). Care was taken to not allow the ground tissue to thaw at any point prior to extraction. This method therefore allowed us to measure gene expression and JA from the same tissue.
Microarray Analysis
To explore a transcriptome-level response of poplar leaves to z3HAC and herbivory, we conducted microarray analysis of the samples collected after 24 hours of herbivore damage.  This allowed a comparison of transcriptome-level responses to herbivory in z3HAC-treated versus untreated leaves (i.e., the set of genes that were primed by z3HAC exposure).  Total RNA was labeled using Ambion's Amino Allyl Message Amp II aRNA Amplification Kit (#AM1753) according to the manufacturer's instructions.  Briefly, 1 ug of each sample was reversed transcribed using T7-oligo(dT) priming and subsequently second strand cDNA synthesis was performed. The resulting double stranded cDNA was purified and then used as a template for in vitro transcription during which amino allyl-modified UTP was incorporated. The resulting amino allyl-modified cRNA was purified and coupled with Cy3 or Cy5 (GE Amersham, #RPN5661) as appropriate. We used dye swapping to account for potential dye effects.  Dye coupled cRNA was purified and 4 ug was fragmented and subsequently dissolved in 60ul of hybridization solution. We used an aspen 7K EST array containing replicate subarrays of 6,705 elements in a 4×4 grid with a spot diameter of ~150 µm and a spacing of 200 µm (Ranjan et al., 2004).  The elements represented ~5,400 unique JGI Populus gene models, and included positive and negative controls (Lucidea Universal ScoreCard, Amersham) to monitor target labeling and hybridization efficiency, as well as many genes of known function. Clone information can be downloaded from the aspenDB website (www.aspenDB.mtu.edu).  Arrays were hybridized following previously published methods (Xiang et al., 2002; Hughes et al., 2001).
Chang S, Puryear J, Cairney J. 1993. A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter 11: 113-116.
Davis JM, Gordon MP, Smit BA. 1991. Assimilate movement dictates remote sites of wound-induced gene expression in poplar leaves. Proceedings of the National Academy of Sciences (USA) 88: 2393-2396.
Frost CJ, Appel HM, Carlson JE, De Moraes CM, Mescher MC, Schultz JC. 2007. Within-plant signalling via volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores. Ecology Letters 10: 490-498.
Hughes TR, Mao M, Jones AR, Burchard J, Marton MJ, Shannon KW, Lefkowitz SM, Ziman M, Schelter JM, Meyer MR, Kobayashi S, Davis C, Dai HY, He YDD, Stephaniants SB, Cavet G, Walker WL, West A, Coffey E, Shoemaker DD, Stoughton R, Blanchard AP, Friend SH, Linsley PS. 2001. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nature Biotechnology 19: 342-347.
Xiang CC, Kozhich OA, Chen M, Inman JM, Phan QN, Chen YD, Brownstein MJ. 2002. Amine-modified random primers to label probes for DNA microarrays. Nature Biotechnology 20: 738-742.

last: Frost

first: Mark

last: Mescher

first: John

last: Davis

last: Carlson

first: Consuelo

last: De Moraes


title: Effects of Mediator subunit gene-deletions under conditions that repress ribosome synthesis

status: Public on Jun 01 2008

city: Bronx

first: Ian

middle: M.

last: Willis

state: NY

address: 1300 Morris Park Ave

department: Department of Biochemistry

institute: Albert Einstein College of Medicine

summary:
Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol) III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally-expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL) genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP) genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription following treatments with rapamycin, chlorpromazine and tunicamycin and in post-diauxic cells. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis.
Keywords: genetic modification, stress response

overall_design: We generated 12 microarray profiles from fluor-reversed replicates of wild-type and med20[delta] strains with or without treatment with rapamycin and under 5 other conditions that repress ribosomal protein gene transcription. The effect of rapamycin on strains deleted for 3 other Mediator subunits was also assessed relative to wild-type.  See Willis et al., (2008) in revision.

first: Gordon

last: Chua

first: Amy

middle: H

last: Tong

first: Renee

last: Brost

first: Timothy

last: Hughes

first: Charles

last: Boone

first: Robyn

last: Moir


title: Response of Lactococcus lactis to glucose starvation

status: Public on Feb 23 2008

city: Toulouse

first: Muriel

last: Cocaign-Bousquet

address: 135 avenue de Rangueil

department: UMR5504 INSA/CNRS - UMR792 INSA/INRA

country: France

institute: Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés

summary:
Adaptation of Lactococcus lactis towards progressive carbon starvation is mediated by three different types of transcriptomic responses: i) global responses, i.e. general decrease of functions linked to bacterial growth and lack of induction of the general stress response, ii) specific responses functionally related to glucose exhaustion, i.e. under expression of central metabolism genes, induction of alternative sugars transport and metabolism genes, induction of arginine deiminase pathway genes and iii) other responses never described previously during carbon starvation.
Keywords: stress response, time course

overall_design: Glucose starvation was imposed by the consumption of this nutrient during the growth of Lactococcus lactis IL1403 on CDM medium and under controlled conditions (30 °C, pH 6.6, nitrogen atmosphere). Cell samples were harvested in exponential and deceleration phases and after 1.25 h and 3.5 h of glucose starvation. Total RNA was extracted from these samples and radiolabelled cDNA were prepared and hybridized on nylon arrays. 2053 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. The 4 time-points were analyzed simultaneously and 3 independent repetitions were performed.

first: Emma

last: Redon

first: Pascal

last: Loubiere


title: High Mobility Group protein HMGB2 is a Critical Regulator of Plasmodium Oocyst Development

status: Public on Apr 19 2008

city: Tampa

first: Kami

last: Kim

address: 3720 Spectrum Blvd, Suite 304

department: Internal Medicine Medicine

institute: University of South Florida

summary:
Two mice per group were infected with one million purified schizonts from the WT and two clones (B3 and D1) of the pyhmgb2 KO lines. Parasitemia reached 10 to 20 % and equivalent gametocytemia total RNA was extracted from the WT and the B3 and D1 clones of the KO parasite lines using the Trizol method on saponin lysed of parasites.
Keywords: gene expression

overall_design: Four samples analysed including dye-swap

first: Mathieu

last: Gissot

first: Li-Min

last: Ting

first: Thomas

last: Daly

first: Lawrence

middle: W

last: Bergman

first: Photini

last: Sinnis


title: Histone turnover mediates epigenetic processes in Drosophila S2 cells

status: Public on Dec 01 2007

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary:
Histone H3 is assembled into nucleosomes during replication but is replaced by the H3.3 variant at sites of active transcription. Thus replacement levels provide a quantitative measure of histone turnover during transcriptional elongation and other active processes in dividing cells. Here we provide new insights into epigenetic regulation with the first genome-wide chromatin profile at 100-bp resolution, based on measurements of H3.3 levels throughout the Drosophila genome.  These data are unpublished.
Keywords: Chromatin affinity-purification on microarray

overall_design: Chromatin affinity profiling of histones H3 and H3.3 on Drosophila tiling arrays

first: Yoshiko

last: Mito

first: Steven


title: comparison of HL60-NG cell and HL60-RG cell

status: Public on Jan 04 2008

city: Shanghai

first: Luan

last: Yang

address: 555 Zu Chong Zhi Road , Zhang Jiang Hi-Tech Park, Pudong,

country: China

institute: Shanghai Institute of Materia Medica , Chinese Academy of Sciences

type:
Genome variation profiling by SNP array
SNP genotyping by SNP array

summary:
Human Promyelocytic Leukemia Cell Line HL60 has been used extensively as a model for myeloid differentiation and leukemia. HL60-RG cell is its sub-line and shows a higher growth rate. In this study, we carried out a genome scan using SNP 10k mapping array on both cells. Comparative study on chromosomal changes of these cells will give us information on mechanism of tumor progression.
Keywords: cell type comparison

overall_design: Two samples HL60-NG cell and HL60-RG cel were analyzed by SNP 10k mapping array.

first: Yang

last: Luan

first: Mieko

last: Kogi

first: Takayoshi

last: Suzuki


title: Mouse embryo or newborn liver or E15.5 embryo minus liver vs. pool of all samples

city: Philadelphia

first: Joshua

last: Friedman

state: PA

address: ARC 1007B, 3615 Civic Center Blvd.

department: Pediatrics

institute: University of Pennsylvania School of Medicine

summary:
Transcriptional profiling of miRNA in embryonic and newborn mouse liver
Keywords: miRNA, liver, newborn, embryonic

overall_design: Time course experiment with common control. Three biological replicates per time point.


title: Caveat Mutator

city: Birmingham

first: Chrystala

last: Constantinidou

department: School of Biosciences

country: United Kingdom

institute: University of Birmingham

type:
Expression profiling by array
Genome variation profiling by array

summary:
The use of whole genome microarrays for monitoring mutagenised or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in Escherichia coli K-12 MG1655 we identified unintended secondary genomic deletions in the flhDC region in deltafnr, deltacrp, and deltacreB mutants.  These deletions were confirmed by PCR and phenotypic tests. Our findings show that non-motile progeny are found in some populations of MG1655 directed deletion mutants, and studies on the effects of gene knock-outs should be viewed with caution when the mutants have not been screened for the presence of secondary deletions, or confirmed by other methods.
Keywords: comparative genomic hybridisation

overall_design: We used the CGH method to genetically characterize a series of regulatory gene deletion mutants we had made in MG1655 using the lamda-Red method of Datsenko and Wanner.  A number of strains were tested using CGH, and each strain was tested only once.  Genomic DNA isolated from wt MG1655 was used as a reference in all hybridisations.

first: Jon

last: Hobman

first: Mala

last: Patel

first: Aida

last: Hidalgo-Arroyo

first: James

last: Cariss

first: Mathew

middle: B

last: Avison

first: Charles

last: Penn

first: Chrystal


title: Zebrafish embryos: Control vs. 5-aza-2'-deoxycytidine treated

status: Public on Oct 14 2009

city: St. John's

first: Tamara

middle: Heather Lorraine

last: Smith

address: Memorial University of Newfoundland

department: Biochemistry

country: Canada

institute: Memorial University

summary: Transcriptional profiling of 24 hours post-fertilization zebrafish embryos comparing control untreated embryos with embryos raised in 75uM 5-azadC. 5-azadC blocks methylation. The goal was to determine genes regulated by methylation during development.

overall_design: Two-condition experiment, 24 hour untreated embryos vs. 5azadC-treated embryos. Biological replicates: 3 control replicates, 3 5azadC-treated replicates.

first: Ross

last: McGowan


title: Expression profile of peripheral blood of healthy individuals phenotyped  based on “Prakriti” concept of Ayurveda

status: Public on Jan 16 2008

city: New Delhi

first: Mitali

last: Mukerji

state: New Delhi

address: Sukhdev Vihar, Mathura Road

department: Functional Genomics and Molecular Medicine

country: India

institute: CSIR-Institute Of Genomics and Integrative Biology

summary:
Immense phenotypic variability exists among healthy individuals which is a consequence of both genetic and environmental effects. Genome wide expression variability is a powerful tool for capturing these expression variations and hence can give insights into genotype phenotype correlations. In the ancient Indian system of medicine, of Ayurveda, the basic tenet for treatment relies on phenotypic assessment In this system, assessment of predisposition,  susceptibility or prognosis for  disease is based on the phenotypic assessment of the individual. Each individuals has a basic constitution which is termed as Prakriti, majorly decided by the relative concentration of three constituents called doshas - Vata (kinetic) Pitta (metabolic) and Kapha (structural) at the time of fertilization. Phenotypically, depending on the concentration the  normal individuals can be classified onto 7 basic types of which 3 are more distinct and are called as Pure Prakriti and the rest of the 4 groups are of mixed types. Pure Prakriti individuals have one of the doshas in more prominence than the other. In this study individuals of Pure Prakrities  have been  selected for elucidation of the transcriptomic variation  in peripheral whole blood cells.
Keywords: Gene expression variability within healthy individuals, subgrouped on the basis of Prakriti (basic constitution of an individual based on Ayurveda)

overall_design: Healthy individuals of pure prakriti (as described in Ayurveda) of age between 18-40 years were identified,  using a questionnaire (for objective assessment) developed, by qualified Ayurvedic practitioners. The individuals have been primarily assessed for phenotypic difference based on morphological characteristics / physiology along with some subjective assessment of physical capabilities and aptitudes. The individuals have been  classified as “Vata” (V), “Pitta” (P) and “Kapha”(K) prakriti. The individuals were provided a similar diet 3 hours prior to sample collection with no interim intake of food or smoking  in between.  In order to find gene expression differences between V, P and K cDNA microarray experiment was performed in pooled samples  wherein males and females experiments were carried out separately. We performed  loop design experiments , where each prakriti was compared with the other two. Technical replicates involving dye swap for each prakriti was used in the same experiment.  We present results of  4 biological replicates and 4 technical replicates of each prakriti in females, and 3 biological and 3 technical replicates of each prakriti in males. Total RNA was isolated from whole blood cells of volunteers using EZ-RNA isolation kit (Biological Industries) as per manufacturers protocol. Double stranded cDNA was synthesized from 15 µg (5 µg each sample) of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each pool of Vata, Pitta and Kapha purified cDNA was divided into two halves, one half labeled with Cy5 and other with Cy3  (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche) and labeled products were purified by Microarray Target Purification Kit (Roche). The two cRNA samples of each biological replicate, one labeled with Cy3 and another with Cy5, were pooled together, precipitated, washed and air-dried. The dried pellet was dissolved in  RNAase free water (Sigma). Hybridization solution was prepared by mixing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma) and added to the labeled product. This mixture was denatured at 65ºC and applied onto cDNA microarray slides. Inter prakriti cohybridization was carried out at 37ºC for 16 hrs. The 48 × 20 × 20 (19,200 spots) human single spot 19K cDNA microarray was procured from University Health Network Microarray Centre, Toronto, Canada. Coverslips were removed by submerging the slides in a solution containing 1X SSC and 0.1% SDS at 50ºC. Slides were washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Finally, slides were washed in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Microarray slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices), using both green and red lasers. The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Analysis was carried out taking two experiments at a time, separately for male and female data. Background subtracted mean values were used for the analysis. All the negative values were removed and only those genes having positive values for all the samples were considered for further analysis.  Values were log2 transformed and lowess normalization was applied to remove dye bias. Across array normalization was carried out using quantile normalization method to correct for systematic variation between arrays. One-way analysis of variance was adopted to identify differentially expressed genes in all three categories. F-test was carried out in order to reject the hypothesis of equality of three group means. in order to investigate further the nature of differentially expressed genes; we have drawn strip charts and carried out pair wise comparisons using t-test. p-values obtained from ANOVA have been adjusted for multiplicity using Bonferroni method for controlling family wise error rate. Genes at p < 0.1 level of significance were considered to be significantly different in expression between all three groups.

first: Sapna

last: Negi

first: Bhavana

last: Prasher

first: Shilpi

last: Aggarwal

first: Shaila

last: Deshmukh

first: Sudha

last: Purohit

first: Farhan

last: Mohammed


title: Identification of transposon insertion sites using the T7 RNA polymerase promoter

status: Public on Jun 26 2007

city: Groningen

first: Anne

middle: de

last: Jong

address: Nijenborgh 7

department: Molecular Genetics

institute: university of Groningen

summary:
Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput genome-wide technique, Genomic Array Footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in non-reproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library, showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1, showed that both have an equal degree of saturation, but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for survival of zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae.
Keywords: GAF Identification of transposon insertion sites

overall_design: Genomic DNA (> 10 µg) was digested overnight with TaqI, DdeI or AluI and the purified DNA was used for an in vitro transcription reaction with the MEGAscript T7 RNA polymerase kit (Ambion) in a total volume of 20 ul.

first: Jetta

last: Bijlsma


title: Gene expression profiling defines ATP as a key regulator of human dendritic cell functions

status: Public on Mar 01 2008

city: Brussels

first: Frederick

last: Libert

address: 808 route de Lennik

department: IRIBHM

institute: ULB

summary:
Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semi-maturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by ATPγS and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPγS initiated an impressive expression profile compared to that of PGE2 (1125 genes compared to 133 genes respectively) but after 24 h, the number of genes regulated by ATPγS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPγS and PGE2 target genes in MoDCs including colony-stimulating factor-1 (CSF-1), MCP-4/CCL13 chemokine, vascular endothelial growth factor (VEGF-A) and neuropilin-1 (NRP-1). ATPγS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion which are involved in monocyte and DC differentiation. Additionally, ATPγS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL8/MCP-2 and CCL13/MCP-4. Interestingly, VEGF-A – a major angiogenic factor displaying immunosuppressive properties - was secreted by MoDCs in response to ATPγS, ATP or PGE2, alone or in synergy with LPS. Finally flow cytometry experiments have demonstrated that ATPγS, ATP and PGE2 down-regulate NRP-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs.
Our data give an extensive overview of the genes regulated by ATPγS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.
Keywords: Time course, treatment comparison

overall_design:
Immature DCs (106 cells/mL) were stimulated by ATPγS (100 µM) or PGE2 (500 nM) for 2, 6, 12 or 24 h in the complete RPMI medium.
Labeled RNA were hybridized to HEEBO arrays containing on average 44544 human 70mer oligonucleotides (Stanford University, US). The data were obtained from two independent donors.

first: Nathalie

last: Bles

first: Michael

last: Horckmans

first: Pascale

last: Macours

first: Hakim

last: El Housni

first: Frederic

last: Marteau

first: Jean-Marie

last: Boeynaems

first: Didier

last: Communi


title: Chromosome fragility at the expanded GAA/TTC tracks depends on repeat orientation and requires mismatch repair system.

status: Public on May 14 2008

city: Durham

first: Piotr

middle: Andrzej

last: Mieczkowski

address: CARL Building

department: Molecular Genetics and Microbiology

institute: Duke University Medical Center

summary:
Expansion of triplex-forming GAA/TTC repeats in the first intron of FRDA gene is known to cause Friedreich’s ataxia.  Besides FRDA, there are a number of other highly polymorphic GAA/TTC loci in the human genome where the size variations so far were considered to be a neutral event.  Using yeast as a model system, we demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements.  The fragility potential strongly depends on the length of the track and orientation of the repeats relative to the replication origin which correlates with their propensity to adopt secondary structure and to block replication progression.  We show that fragility is mediated by mismatch repair machinery and requires the MutS(beta) and endonuclease activity of MutL(alpha).  We suggest that the mechanism of GAA/TTC-induced chromosome aberrations defined in yeast can also operate in human carriers with expanded tracks.
Keywords: CGH-array

overall_design: Genomic DNA from each of 15 strains was competitively hybridized to DNA from the parent diploid strain (Cy3/green). Gains of genomic segments in the survivors were detected as continuous regions of positive Log2 Red:Green ratios, while losses were detected as negative Log2 Red:Green ratios.

first: Hyun-Min

last: Kim

first: Vidhya

last: Narayanan

first: Mieczkowski

middle: A

last: Piotr

first: Thomas

last: Petes

first: Sergei

last: Mirkin

first: Kirill

last: Lobachev


title: Gene expression "Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii"

status: Public on May 22 2007

first: Kami

type: Expression profiling by genome tiling array

summary:
total RNA was extracted after 24 hours of development of the RH strain in HFF cells.
Keywords: gene expression

sample_organism:
Homo sapiens
Toxoplasma gondii RH

overall_design: RH strain of Toxoplasma gondii was grown in HFF cells during 24 hours.

first: Mathieu

last: Gissot

first: Krystina

last: Kelly

first: James

last: Ajioka

first: John

last: Greally


title: ChIP on chip "Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii"

summary:
ChIP on chip with modified histones antibodies
Keywords: ChIP-chip

overall_design: RH strain of Toxoplasma gondii was grown in HFF cells during 24 hours and subjected to ChIP using modified histones antibodies


status: Public on Jan 01 2008

city: Houston

first: Molly

last: Bray

address: 1100 Bates, Suite 8070

department: Pediatrics/USDA/ARS Children's Nutrition Research Ctr

institute: Baylor College of Medicine

summary:
Disruption of the Circadian Clock within the Cardiomyocyte Influences Myocardial Contractie Function, Metabolism, and Gene Expression
Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock.  The role of this transcriptionally-based molecular mechanism in cardiovascular biology is poorly understood.  We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variations in myocardial biology.  We therefore generated a cardiomyocyte-specific circadian clock mutant (CCM) mouse, in order to test this hypothesis.  At 12 weeks of age, CCM mice exhibit normal myocardial contractile function in vivo, as assessed by echocardiography.  Radiotelemetry studies reveal attenuation of heart rate diurnal variations and bradycardia in CCM mice (in the absence of conduction system abnormalities).  Reduced heart rate persisted in CCM hearts perfused ex vivo in the working mode, highlighting the intrinsic nature of this phenotype.  Wild-type, but not CCM, hearts exhibited a marked diurnal variation in responsiveness to an elevation in workload (80mmHg plus 1 microM epinephrine) ex vivo, with a greater increase in cardiac power and efficiency during the dark (active) phase versus the light (inactive) phase.  Moreover, myocardial oxygen consumption and fatty acid oxidation rates were increased, while cardiac efficiency was decreased, in CCM hearts.  These observations were associated with no alterations in mitochondrial content or structure, and modest mitochondrial dysfunction, in CCM hearts.  Gene expression microarray analysis identified 548 and 176 genes in atria and ventricles, respectively, whose normal diurnal expression patterns were altered in CCM mice.  These studies suggest that the cardiomyocyte circadian clock influences myocardial contractile function, metabolism, and gene expression.
Keywords: Comparison of circadian oscillations in gene expression in hearts taken from wildtype and transgenic animals

overall_design: Atrial and ventricular tissue samples were collected from wildtype and transgenic animals lacking a functional circadian clock within the cardiomyocytes every three hours for a period of 24 hours (n=4 samples per time point; total of 128 samples).  Circadian patterns of gene expression were fit for each gene in wild-type atria and ventricle, and then differences in oscillating genes between wild-type and transgenic tissues were determined.

first: Chad

last: Shaw

first: Michael

last: Moore

first: Rodrigo

last: Garcia

first: Melissa

last: Zanquetta

first: David

last: Durgan

first: William

last: Jeong

first: Ju-Yun

last: Tsai

first: Heiko

last: Bugger

first: Dongfang

last: Zhang

first: Andreas

last: Rohrwasser

first: Julie

last: Rennison

first: Jason

last: Dyck

first: Sheldon

middle: E

last: Litwin

first: Paul

last: Hardin

first: Chi-Wing

last: Chow

first: Margaret

middle: P

last: Chandler

first: E

last: Abel

first: Martin

last: Young


title: A genome wide role for CHD remodeling factors and Nap1 in nucleosome disassembly

status: Public on May 19 2007

city: Stockholm

first: Karl

last: Ekwall

address: Alfred Nobels Alle 7

country: Sweden

institute: Karolinska Inst

type: Genome binding/occupancy profiling by array

summary:
Chromatin remodeling factors and histone chaperones were previously shown to cooperatively affect nucleosome assembly and disassembly processes in vitro. Here we show that S. pombe CHD remodellers, Hrp1 and Hrp3 physically interact with the histone chaperone Nap1. Genome wide analysis of Hrp1, Hrp3 and Nap1 occupancy, combined with nucleosome density measurements in respective mutants revealed that the CHD factors and Nap1 co-localized in particular to promoter regions where they remove nucleosomes near the transcriptional start site. Hrp1 and Hrp3 also regulate nucleosome density in coding regions where they have redundant roles to stimulate transcription. Previously, DNA replication dependent and independent nucleosome disassembly processes have been described. We found that nucleosome density increased in the hrp1 mutant in the absence of DNA replication. Finally, regions where nucleosome density increased in hrp1, hrp3 and nap1 mutants also showed nucleosome density and histone modification changes in HDAC and HAT mutants. Thus, this study revealed an important in vivo role for CHD remodellers and Nap1 in nucleosome disassembly at promoters and coding regions, which are linked to changes in histone acetylation.
Keywords: ChIP on CHIP and expression profiling

overall_design:
Expression profiling experiments were performed and quantified according to (Xue et al., 2004). Data was normalized using Lowess per spot per chip intensity-dependent Gene Spring v 7.2 (Silicon Genetics).
IGR and ORF DNA microarrays (Eurogentec) and GeneChip® S. pombe Tiling Array (Affymetrix) were used to create ChIP on CHIP protein binding and histone H3 density maps. Cells cultures for both microarray platforms were grown to mid logarithmic phase, harvested, fixated lysed and ChIPed as described in (Sinha et al., 2006). Antibodies specific for a-myc (9E10, Sigma) and anti-H3 C-terminal antibody (AB1791, Abcam) were used for the ChIP.
For the Eurogentec ChIP on CHIP DNA microarrays, 1.0 mg of input DNA and ChIP DNA was labelled with either Cy3 or Cy5 fluorescent dyes respectively and both labelled samples were together hybridized and quantified according to (Wiren et al., 2005). All binding maps and the histone H3 density experiments were performed in at least duplicate experiments generating 6 respective 4 measurement points since each microarray slide contain duplicate spots for each fragment. In addition, we performed cy3 and cy5 dye swap for every experiment to correct for dye bias. HDAC ChIP on CHIP data published in (Wiren et al., 2005), containing acetylation levels and H3 density were quality filtered, normalisednormalized and combined to generate direct effects in histone density changes, as described above for the Hrp1, Hrp3 and Nap1.
The Affymetrix Tiling arrays ChIP DNA was extracted as described above and amplified to 5.0 mg using protocol published in (Robyr and Grunstein, 2003). Next, DNA was fragmentated to ~100 bp using DNAse I, followed by biotin labelling and hybridized according to (Lengronne et al., 2004). Affymetrix Tiling arrays to determine H3 density throughout genes at direct binding for Hrp1 and Hrp3 were performed once.

first: Julian

last: Walfridsson

first: Olga

last: Khorosjutina

first: Claes

middle: M

last: Gustafsson


title: Identification of dysregulated genes in lymphocytes from children with Down syndrome

status: Public on Apr 25 2007

city: Sao Carlos

first: César

middle: Adolfo

last: Sommer

state: Sao Paulo

address: Rodovia Washington Luis (SP-310), km 235

department: Genetics and Evolution

country: Brazil

institute: Universidade Federal de Sao Carlos

summary:
The aim of this study was to analyze global changes of gene expression in lymphocytes from children with trisomy 21 by means of the serial analysis of gene expression (SAGE) methodology. Comparison between DS and normal profiles revealed that most of the transcripts were expressed at similar levels. Among the 242 significantly differentially expressed SAGE tags, many of them corresponded to genes involved in transcription, RNA processing, signaling, immune response and lipid metabolism. Our results indicate that trisomy 21 induces a modest dysregulation of disomic genes that may be related to the immunological perturbations seen in DS.
Keywords: SAGE analysis in normal and Down syndrome lymphocytes

overall_design: Lymphocytes were isolated from peripheral blood of normal and karyotypically confirmed full trisomy 21 children. Children included in the study, ranging in age from 1-4 years, were self reported as free of chronic or acute infections. Total RNA was isolated with TRIzol reagent (Invitrogen). The SAGE libraries were constructed using pooled RNA from six DS children (DS library) and six normal children (control group) to eliminate any individual variation. SAGE was performed by means of the I-SAGE kit (Invitrogen) following the manufacturer’s instructions.

first: C

first: A

middle: C

last: Pavarino-Bertelli

last: Goloni-Bertollo

first: F

last: Henrique-Silva


title: proteomic survey of lung development and nmyc function in the lung

status: Public on Apr 24 2007

city: Toronto

first: Brian

middle: Joseph

last: Cox

state: Ontario

address: 1 King's College Circle, Rm 3360

department: Physiology

institute: University of Toronto

summary:
protein profiling of mouse lung organogenesis from embryonic day E13.5 until adulthood by gel-free two-dimensional liquid chromatography coupled to large-scale tandem mass spectrometry (MudPIT).

protein expression in Nmyc loss of function mutant with a comparison to wildtype at E18
Keywords: protein, proteomics, Mudpit, lung, development, nuclear, cytosol, mitochondria, mouse

overall_design:
Mouse tissues
Mouse tissues were derived from ICR timed mated mice. Timing of conception was designated by assigning noon of the day of plug formation as day 0.5. Pregnant animals were sacrificed and uteri dissected to obtain individual embryos from which lung tissue was derived. A minimum of 0.5 grams or more lung tissue was used in all cases, which required approximately 50 pairs of lungs at E13.5, 24 at E16.5, and at later stages we homogenized lungs from a minimum of 2 litters (~24 pairs of lungs) but isolated protein extracts from an aliquot of the homogenate equivalent to 4 grams of lung tissue. Nmyc mice heterozygous for the hypomorphic allele were timed mated and litters delivered on Day 18.5. The uterus was removed and the pups dissected from the uterus. The yolk sac and amnion were carefully removed. The placental vein and artery were left intact while the pup was dried with Kim wipes, then they were cut and the pups moved under a heat lamp for warmth. The pups mouth and nose were frequently blotted to remove any mucus that was expelled. They were also gently rubbed to stimulate breathing. After 30 minutes pups that had failed to breath or had become listless were sacrificed, a tail clipping removed for genotyping as described in [1] and lungs removed and processed for protein. Geneotyping was done using the following primer pairs: for Nmyc wild type allele NmycA GGT AGT CGC GCT AGT AAG AGC and NmycB GGC GTG GGC AGC AGC TCA AAC and for Nmyc 9a hypomorphic allele NmycB and NmycC (neo) GGA GAA CCT GCG TGC AAT CC.

Tissue fractionation
The tissue fractionation was done as described [2]. Briefly, lung tissue was rinsed twice in ice cold phosphate-buffered saline and homogenized in ice cold lysis buffer, containing 250 mM sucrose, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM DDT, and 1 mM PMSF using a tight fitting Teflon pestle. The lysate was centrifuged in a bench-top centrifuge at 800 g for 15 minutes; the supernatant served as source for cytosol, mitochondria and microsomes. The pellet, which contains the nuclei was re-homogenized in lysis buffer and centrifuged as above. The nuclei were resuspended in 2 molar sucrose buffer (2M sucrose, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM DDT, and 1 mM PMSF) and pelleted by ultracentrifugation at 80000 g in a SW40Ti (Beckman) for 35 minutes. Mitochondria were isolated from the crude cytoplasmic fraction by bench-top centrifugation at 6000 g for 15 minutes. The mitochondrial pellet was washed and pelleted twice in lysis buffer. The cytosolic fraction was obtained after removal of the microsomal fraction by ultracentrifugation at 100000 g in a SW60Ti (Beckman) for 1 hour.

Organelle protein extraction
Nuclear proteins were extracted by resuspending the isolated nuclei in 5 volumes of 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, and 25 % glycerol for 30 minutes with gentle shaking at 4ºC. The nuclei were lysed by 10 passages through an 18-gauge needle, and debris was removed by microcentrifugation at 13000 rpm. The supernatant served as nuclear fraction. Mitochondrial proteins were isolated by incubating the mitochondria in a hypotonic lysis buffer [10 mM HEPES, pH 7.9] for 30 minutes on ice, briefly sonicated and debris pelleted at 13000 rpm for 30 minutes.

Protein digestion and MudPIT analysis
An aliquot of 150 ug of total protein from each fraction was precipitated over night with 5 volumes of ice-cold acetone followed by centrifugation at 13000 rpm for 20 minutes. The protein pellet was solubilized in 8 M urea, 50 mM Tris-HCl (pH 8.5), 1 mM DTT for 1 hour followed by carboxyamidomethylation with 5 mM iodoacetamide for 1 hour at 37ºC. The samples were then diluted to 4 M urea with 100 mM ammonium bicarbonate (pH 8.5) and digested over night with endoproteinase Lys-C at 37ºC. The next day the mixture was further diluted to 2 M urea with 50 mM ammonium bicarbonate (pH 8.5) supplemented with CaCl2 to a final concentration of 1 mM, and incubated over night with Porozyme trypsin beads at 30ºC with rotating. The resulting peptide mixture was solid phase-extracted with SPEC-Plus C18 cartridges according to the manufacture’s instruction and stored at -80ºC until further use.

Mass spectrometry
A fully automated 12-cycle, 24 hour MudPIT chromatographic procedure was set up essentially as described previously [3]. Briefly, an HPLC quaternary pump was interfaced with an LCQ DECA XP ion trap tandem mass spectrometer (ThermoFinnigan, San Jose, CA). A 100 um inner diameter fused silica capillary microcolumn (Polymicro Technologies, Phoenix, AZ) was pulled to a fine tip using a P-2000 laser puller (Sutter Instruments, Novato, CA) and packed with 10 cm of 5-um Zorbax Eclipse XDB-C18 resin (Agilent Technologies, Mississagua, Ontario, Canada) and then with 6 cm of 5-um Partisphere strong cation exchange resin (Whatman). Samples were loaded manually onto separate columns using a pressure vessel. The chromatography was carried out as described by Wolters et al. [4].
Protein identification and Validation
The SEQUEST program (a kind gift from Jimmy Eng and John Yates III) was used to search peptide spectra essentially as described previously using a minimally redundant mouse/human protein sequence database (EBI database) [5, 6]. The statistical confidence of identified proteins was validated by the use of an in-house algorithm (STATQUEST as described earlier [6].
Quantitative analysis
The profiles were clustered based on the Spearman rank correlation metric with average linkage using the freeware program Cluster 3.0 based on the original Cluster program [7]. The resulting clusters were visualized in heat-map format using Java TreeView based on the original TreeView [7]. Spectral counts were normalized essentially as described [8]. Relative protein abundance was inferred using the normalized spectral counts as a semi-quantitative metric after arcSine(H) transformation of the data.

1. Moens, C.B., et al., A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Dev, 1992. 6(5): p. 691-704.
2. Kislinger, T., et al., Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling. Cell, 2006. 125(1): p. 173-86.
3. Washburn, M.P., D. Wolters, and J.R. Yates, 3rd, Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol, 2001. 19(3): p. 242-7.
4. Wolters, D.A., M.P. Washburn, and J.R. Yates, 3rd, An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem, 2001. 73(23): p. 5683-90.
5. Eng, J.K., A.L. McCormack, and J.R. Yates III, An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry, 1994. 5(11): p. 976-89.
6. Kislinger, T., et al., PRISM, a Generic Large Scale Proteomic Investigation Strategy for Mammals. Mol Cell Proteomics, 2003. 2(2): p. 96-106.
7. Eisen, M.B., et al., Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A, 1998. 95(25): p. 14863-8.
8. Cox, B., T. Kislinger, and A. Emili, Integrating gene and protein expression data: pattern analysis and profile mining. Methods, 2005. 35(3): p. 303-14.

middle: J

first: Thomas

last: Kislinger

first: Andrew

last: Emili

first: Janet

last: Rossant


title: Tissue Contaminants and Associated Transcriptional Response in Trout Liver from High Elevation Lakes of Washington

status: Public on May 01 2007

city: Woods Hole

first: Neelakanteswar

last: Aluru

state: MA

address: 45 Water Street

department: Biology

institute: Woods Hole Oceanographic Institution

summary:
The consistent cold temperatures and large amount of precipitation in the Olympic and Cascade ranges of Washington State are thought to increase atmospheric deposition of contaminants in these high elevation locations. Total mercury and 28 organochlorine compounds were measured in composite, whole fish samples collected from 14 remote lakes in the Olympic, Mt. Rainer, and North Cascades National Parks.  Mercury was detected in fish from all lakes sampled and ranged in concentration from 17 to 262 ug/kg wet weight. Only two organochlorines, total polychlorinated biphenyls (tPCB) and dichlorodiphenyldichloroethylene (DDE), were detected in fish tissues (concentrations <25 ug/kg wet weight).  No organochlorines were detected in sediments (MRL ≈1-5 ug/kg), while median total and methyl mercury in sediments were 30.4 and 0.34 ug/kg (dry weight), respectively.  Using a targeted  rainbow trout cDNA microarray with known genes, we detected significant differences in liver transcriptional responses, including metabolic, endocrine, and immune-related genes, in fish collected from a contaminated lake compared to a lake with a lower contaminant load.  Overall, our results suggest that local urban areas are contributing to the observed contaminant patterns, while the transcriptional changes point to a biological response associated with exposure to these contaminants in fish. Specifically, the gene expression pattern leads us to hypothesize a role for mercury in disrupting the metabolic and reproductive pathways in fish from high elevation lakes in western Washington.
Keywords: High altitude lakes, mercury, salmonids, organochlorines

sample_organism:
Oncorhynchus mykiss
Oncorhynchus clarkii

overall_design: Overall, 5 independent samples from Wilcox lake and 3 independent samples from Skymo lake were used for microarray. All the 8 samples were cross hybridized using reference RNA obtained from Rainbow trout liver samples. Dye-swapping was also followed.

first: Patrick

last: Moran

first: Robert

last: Black

first: Mathilakath

last: Vijayan


title: The role of multi-copper oxidase CueO

status: Public on Apr 28 2007

first: Chrystala

last: Constantinidou

summary:
The effect of a cueO mutation on global gene expression in E. coli has been analysed using whole genome oligonucleotide microarrays. A cueO mutant lacks a periplasmic multi-copper oxidase, an enzyme that is believed to be involved in the oxidation of cuprous and ferrous ions as well as phenolic compounds in the periplasm. Deletion renders the cell sensitive to copper and this toxicity is likely associated with increased oxidative stress in a cueO mutant.
Keywords: genetic modification

overall_design: Triplicate cultures of E. coli MG1655 and the isogenic cueO mutant were grown to mid-exponential phase (OD600 0.8) in M9C supplemented with 2uM FeSO4. RNA was extracted using Qiagen RNAprotect and RNeasy Mini Kit. Reference RNA was similarly purified from triplicate cultures of E. coli MG1655 grown on M9C supplemented with 2uM FeCl3. RNA concentration and quality was assessed using an Aligent Bioanalyser 2100 NanoLabchip.

first: Jai

last: Tree

first: Glenn

last: Ulett

first: Jon

middle: L

last: Hobman

first: Nigel

last: Brown

first: Christopher

last: Kershaw

first: Mark

last: Schembri

first: Michael

last: Jennings

first: Alastair

middle: G

last: McEwan


title: Non-coding RNA tiling array in rice

status: Public on Feb 06 2007

city: New Haven

first: Xiangfeng

last: Wang

state: CT

address: 352 OML, 165 Prospect st

department: MCDB

institute: Yale University

summary:
The 1 Mb tiling array was used to represent the 10.0 Mb to 11.0 Mb region of japonica rice Chromosome 10. A tile path was designed with 36-mer probes at a step of 5 nt. All 388,994 probes were retained and synthesized into a single array. This array was hybridized to the cDNA target used for the full-genome tiling array as well as targets prepared from non-coding RNA sample I and II.
Keywords: noncoding RNA

overall_design: Non-coding RNA species were isolated from total RNA as previously described (5). Briefly, total RNA isolated from japonica rice leaves using the Trizol method was loaded onto an Qiagen-tip and eluted with 0.6~1.0M NaCl gradient of QRW2 buffer (Qiagen) at 50 oC. Fractions corresponding to tRNAs (sample I) and small RNAs of 80-500 nucleotides (nt) (sample II) were collected. To remove remaining mRNAs and rRNAs, the small RNA fractions were hybridized to a mixture of specifically designed oligos in the binding buffer. Unwanted RNA molecules were targeted and removed by a magnetic bead based process (MicrobExpress kit, Ambion). The enriched non-coding RNA pool was then dephosphorylated with a calf intestine alkaline phosphatase and ligated to a 3' adaptor oligonucleotide by the T4 RNA ligase (5). Small size molecules and excessive adaptors were removed from the ligation products using the mirVana miRNA isolation kit (Ambion) and reverse transcribed at 50 oC using the Thermoscript (Invitrogen) and the oligo 3RT as the primer (5). Amino-allyl-modified dCTP was incorporated into the cDNA strand during reverse transcription.


title: TGFbeta- and IMR induced epithelial cell differentiation in a three-dimensional intestinal crypt-villus axis model

status: Public on Jun 27 2007

city: Tampere

first: Kati

middle: Marjaana

last: Juuti-Uusitalo

address: Biokatu 10

department: Medical School

country: Finland

institute: Pediatric Research Center

summary:
The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFbeta1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model differentiation seen in jejunal crypt-villus axis (Halttunen et al. 1996). The gene expression changes were studied in fully undifferentiated and fully differentiated three-dimensional T84 cultures with cDNA microarray method.
Keywords: developmental stage

overall_design:
Human intestinal epithelial T84 cells (CCL 2´48, ATCC Rockville, MD, USA) were cultured in three-dimensional type I collagen gel as previously described Halttunen et al 1996 . T84 cells were induced to differentiate either by adding 20 ng/ml human recombinant TGFbeta1 (hTGF-beta1, R&D Systems Europe, Oxon, UK) or by soluble factors secreted by IMR-90 type human embryonic lung fibroblasts (CCL 186, ATCC). IMR fibroblasts, cultured on the top of the epithelial cells, were separated from the epithelial cells by cell-free collagen layer. T84 cells cultured within collagen gel supplemented with medium were used as undifferentiated control. We studied only fully undifferentiated and fully differentiated epithelial cells harvested after seven days of culturing. All experiments were carried out in triplicate.
Isolation of RNA:The mRNA was extracted from the cell culture samples to ice-cold TRIzol reagent (Life Technologies, Inc. Frederick, MD, USA) according to the manufacturer’s protocol. All samples were subjected to DNAse I treatment (Roche Diagnostics GmbH, Mannheim, Germany). Total RNA was quantitated by spectrophotometry and quality checked by agarose gel electrophoresis.
cDNA synthesis and array hybridization
Gene expression was monitored using a Human GeneFilter GF200 (Research Genetics, Huntsville, AL, USA), consisting of 5188 test sequences, 96 control points and 192 housekeeping genes. Arrayed sequences contained both genes with known or predicted function and expressed sequence tags (ESTs) with unknown function. Probe preparation and microarray hybridization were performed following manufacturer’s (Research Genetics) protocol using 1.5 microg total RNA as template, 10microl (10mCi/ml) 33P dCTP (ICN Radiochemicals), 1,5microl dNTP mix containing dATP, dTTP, dGTP at 20 mM (Pharmacia ), 1,5 microl reverse transcriptase (Supercript II, Life Technologies), 1,0 microl DTT. Elongation for 90 min at 37oC. The label was purified with Bio-Spin 6 Chromatography column (Bio-Rad), and hybridized for 12 h at 42oC in roller bottle. After hybridization membrane was washed twice in 2 X SSC 1%SDS, 0.5XSSC. The hybridized membrane was let to  expose Phosphoimager screens for 12 h. Hybridization signals were detected on a Storm 860 phosphoimager (Molecular Dynamics, Amersham Biosciences, Buckinghamshire, England) .
Microarray data analysis:Filter images were aligned and spot intensities analysed with Pathways Software (Research Genetics). Raw data were imported to Microsoft Excel. The handling of the raw data, normalization and improvement of fidelity by setting a cut-off value were done as previously described in Juuti-Uusitalo et al. 2004. Intensity values were normalised in order to avoid possible differences in the amounts of RNA and differences in hybridisation efficiency. The normalisation factor was determined before background subtraction. The normalisation was done by the sum method (Kroll et al. 2002). The scaling factor was set to ensure that the sums of spot intensities were equal for all filters.

first: Katri

last: Kaukinen

first: Markku

last: Mäki

first: Heikki

last: Kainulainen


title: MPSS profiling of embryonic gonad and primordial germ cells

status: Public on Mar 01 2007

city: Seoul

first: heebal

last: kim

address: San 56-1 Sillim-dong Gwanak-gu

country: South Korea

institute: Seoul National University

summary:
We used the MPSS technology to uncover gene expression profiling in a greater depth in the early embryonic gonads and primordial germ cells (PGCs) in the chicken. Total numbers of sequenced signatures were 1,012,533 and 995,676 for the PGCs and gonad. Using a false discovery rate cut-off of 0.05, we found 3.05 % of all signatures in the PCGs and 1.89 % of all signatures in the gonad signatures were significantly up-regulated compared to each sample. The MPSS result was very consistent with our previous result using EST data(http://chickgce.snu.ac.kr/).
Keywords: cell type comparison

overall_design:
Experimental animals provided for this experiment were maintained at the University Animal Farm, Seoul National University, and all experimental procedures were performed at the affiliated laboratories of the university. Gonadal cells were retrieved from the gonads of 6.5-day-old (stage 29) White Leghorn (WL) embryos by our standard procedure [26]. Embryos were freed from the yolk by rinsing with calcium- and magnesium-free PBS and the gonads were retrieved by dissection of embryo abdomen with sharp tweezers under a stereomicroscope. Embryonic gonads were collected from total 1,947 embryos by 10 highly-skilled persons through 8 separated experimental batches. Gonadal tissues were dissociated by gentle pipetting in 0.05% (v:v) trypsin solution supplemented with 0.53 mM EDTA. After centrifuged at 200xg for 5 min, total gonadal cells were loaded into MACS (Miltenyi Biotech, Germany), and the separated primordial germ cells (PGCs) were immediately stored in liquid nitrogen (-190°C) until processed further. The numbers of PGCs in cell population before and after loading were counted.

MACS treatment for chicken PGCs and counting PGC number
Chicken gonadal cells were incubated with PGC-specific primary antibody, anti-stage specific embryo antigen (anti-SSEA)-1 antibody for chicken PGCs (mouse IgM isotype), for 20 min at the room temperature of 20-25°C. Anti-SSEA-1 antibody developed by [27] was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Development of Biological Science. After washing with 1mL buffer (PBS supplement with 0.5% BSA and 2mM EDTA), the supernatant was completely removed. The pellet was mixed with 100 µl of buffer containing 20 µl of rat anti-mouse IgM microbeads for 15 min at 4°C. Treated cells were carefully washed by the addition of 500 µl of buffer and subsequently loaded with MACS.[28] For counting cell number, chicken PGCs before or after MACS treatment were fixed with 1% (v:v) glutaraldehyde for 5 min and rinsed with 1x PBS twice. The anti-SSEA-1 ascites fluid diluted 1:1,000 in PBS was added and subsequent steps were carried out using DAKO universal LSABÂ® kit, Peroxidase (DAKO, USA) according to the manufacturer's instruction.
After 8 batches of cell preparation, total cell number of PGC-enriched fraction and gonadal stromal cells was 5.26X106 and 1.76X108, respectively. These cell populations were further used for total RNA isolation and massively parallel signature sequencing (MPSS) analysis.

first: Heebal

last: Kim

first: Tae

middle: S

last: Park

first: Woon

middle: K

last: Lee

first: Sunjin

last: Moon

first: Jin

middle: N

first: Ji

middle: H

last: Shin

last: Jung

first: Seon

middle: D

first: Sang

first: Kyung

first: Mi

first: Mina

last: Jeon

first: Jungrye

last: Nam

first: Yeonkyung

last: Kang

first: Jeong

last: Lim

first: Jae

middle: Y

last: Han


title: Gene expression in endometrial cancer cells (Ishikawa) after short time high dose exposure to progestins

status: Public on Apr 13 2007

city: Tromsø

first: Ruth

middle: Hracky

last: Paulssen

address: MH-Bygget Breivika

department: Institute of Clinical Medicine

country: Norway

institute: Universitetet i Tromsø

summary:
Gene expression in endometrial cancer cells (Ishikawa) after short time exposure to progestins
Sample and RNA preparation:
The series is composed of sixteen hybridizations for analysis of differentially expressed genes in endometrial cancer cells (Ishikawa). In eight independent experiments, Ishikawa cells in monolayer received with 30 ug/ml progestin and were grown in RPMI-1640 medium (Sigma, Sty. Louis) with 10% dialyzed foetal calf serum (PAA-laboratories, Austria) without phenol red and antibiotics. Cells were seeded at a density of 1 x 105 cells/ml in 25 cm2 culture flasks in a humidified atmosphere (5% CO2) at 37°C for 4 hours. Cells not treated with progestin served as control groups. Total RNA extraction was performed using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY).
Labeling and hybridization:
Total RNA was reverse transcribed and labeled with Cy3- and Cy5- attached dendrimer, respectively, using the Genisphere 3DNA 350HS kit (Genisphere, Montvale, NJ) as described in the manufacturer's protocol. Hybridizations of transcribed probes were carried out in a TECAN HS4800 instrument using the formamide-based hybridization buffer from Genisphere containing 5% dextrane sulfate and 5.5 ng/ul COT1 DNA (GIBCO BRL Life technologies) at 37º for 23 hours. 3DNA dendrimer hybridizations were carried out in formamide-based hybridization buffer alone. Post-hybridization washes were carried out at room temperature with 2xSSC for 1 min, 0.2% SDS /2xSSC for 1 min and finally with 0.2xSSC for 30 sec. Sixteen hybridizations including dye-swap have been performed for eight independent experiments.
Scanning, image analysis and data processing:
The arrays were scanned with the GenePix 4000B scanner (Axon Instruments Inc). The features were extracted from the arrays using GenePix Pro 6.0  (Axon instruments Inc., Union City, CA). All subsequent statistical analyses were performed using the statistical package R (R Development Core Team). Spots that displayed a signal-to-noise ratio of less than 2, or that were significantly saturated (more than 20 % saturation among foreground pixels) were filtered out. The median was used as the averaging measure of the foreground pixels.  After quality control, genes that were present in less than 50 % of the arrays were filtered out. The arrays were normalized using dye-swap normalization. One array, whose dye swap twin failed was normalized using the lowess method. Statistical significance was assigned to the genes using the SAM methodology. The bioconductor SAMR package was used in the actual analysis. Missing expression levels were imputed using 10 nearest neighbour's imputation. The local FDR threshold was set to 25 %.

Keywords: Progestin treatment, Control-treatment

overall_design: Eight ishikawa cell samples were treated independently with progestin. Eight untreated samples were used as controls. Each pair of treated-untreated cells was matched by a dye-swapped technical replicate for a total of 32 samples hybridized directly together on 16 arrays.

first: Halvor

last: Grønaas

first: Anne

last: Ørbo