title: Genes associated with alcohol abuse and nicotine addiction in the human nucleus accumbens

city: Brisbane

first: Traute

last: Flatscher-Bader

state: Queensland

address: The University of Queensland, Upland Road, QBI Building (79)

department: The Queensland Brain Institute

institute: The University of Queensland

summary:
Background:  The incidence of alcohol and tobacco co-abuse is as high as 80%.  The molecular mechanism underlying this comorbidity is virtually unknown but interactions between these drugs have important implications for the development of, and recovery from drug dependence.  Methods:  We investigated the effects of chronic tobacco and alcohol abuse and the interaction of the two behaviours on global gene expression in the human nucleus accumbens using cDNA microarrays and 20 alcoholic and control cases, with and without smoking comorbidity.  Changes in gene expression were established by two-way ANOVA.  Unsupervised hierarchical clustering was utilized to probe the strength of the data sets.
Results:  Subjecting the data sets derived from microarray gene expression screening to unsupervised hierarchical clustering tied the cases into distinct groups.  When considering all alcohol-responsive genes, alcoholics were separated from non alcoholics with the exception of one control case.  All smokers were distinguished from non smokers based on similarity in expression of smoking-sensitive genes. In the nucleus accumbens, alcohol-responsive genes were associated with transcription, lipid metabolism and signalling.  Smoking-sensitive genes were predominantly assigned to functional groups concerned with RNA processing and the endoplasmic reticulum.  Both drugs influenced the expression of genes involved in matrix remodelling, proliferation and cell morphogenesis.  Additionally, a gene set encoding proteins involved in the canonical pathway ‘regulation of the actin cytoskeleton’ was induced in response to alcohol and tobacco co-abuse and included.  Conclusions:  The region-specific modulation of alcohol-sensitive gene expression by smoking may have important consequences for alcohol-induced aberrations within the mesolimbic dopaminergic system.

overall_design: The data set contained individual RNA samples extracted from the nucleus accumbens from 5 non smoking non drinking control case, 5 non smoking alcoholics, 5 non drinking smokers, and 5 smoking alcoholics. An aliquot if each sample was combined to generate a reference pool. Each sample was amplified individually and labelled with Cy3 in the process, the reference samples was amplified and labelled with Cy5. Each sample was hybridised competitively against the reference sample to 1 array.

first: Erin

last: Harrison

first: Izuru

last: Matsumoto

first: Peter

last: Wilce


title: Development of a versatile, target-oriented tiling microarray assay for measuring allele-specific gene expression

status: Public on Jul 01 2010

city: New Haven

first: Hang

last: He

state: CT

address: 165 Prospect St

department: MCDB

country: USA

institute: Yale University

summary: In the study of gene expression regulation in plants, it is often desirable to distinguish transcript pools derived from different alleles present in the same organism. We report here an oligonucleotide tiling microarray designed to specifically target 518 single nucleotide polymorphisms (SNPs) between the two sequenced rice (Oryza sativa) subspecies indica and japonica. The tiling array includes all pairs of 25-mer allele-specific probes interrogating each SNP by placing the polymorphic site at all 25 possible positions within the probe. Hybridization of the tiling array to a titration series in which the japonica- and indica-derived cDNA templates are mixed with altering proportions and development of a novel bioinformatic methodology allowed us to screen for diagnostic probe pairs for each SNP. Our result indicates that 284 (55%) SNPs have at least one diagnostic probe pair suitable for distinguishing and quantifying the relative abundance of allele-specific transcripts. As a proof-of-concept, we analyzed allele-specific expression in reciprocal indica × japonica F1 hybrids and detected imbalanced expression at 95 (33%) and 71 (25%) SNPs, respectively. Comparison results from RNA-sequencing and allele-specific real-time PCR experiment validates the sensitivity and reliability of the tiling array method. Together, our results demonstrate the advantages of the tiling array method in interrogating large numbers of SNPs and for the reliability of the experimental and analytical techniques used for quantifying allele-specific gene expression.

overall_design: For each selected SNP, 25 pairs of 25-mer oligonucleotide probes were designed with one match perfectly to the indica allele and the other the japonica allele within each pair. Among the 25 probe pairs, the polymorphic site was placed at a different position such that all possible 25-mer allele-specific probes spanning the polymorphic site were included. The resultant 25,900 probes were synthesized in triplicate in a single microarray produced on the Maskless Array Synthesizer platform as previously described (Li et al., 2005; 2006). Poly(A+) RNA from japonica, indica and their reciprocal hybrids was reverse transcribed using an oligo(dT)18 primer, during which amino-allyl-modified dUTP (aa-dUTP) was incorporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare) to the amino-allyl functional groups in the cDNA. Dye-labeled target was quantified using a spectrophotometer and two μg used for hybridization to each array. In the titration series experiment, the dye-labeled targets from japonica and indica were mixed in known proportions before hybridization.

first: He

last: Hang

first: Zhang

last: Huiyong

first: Wang

last: Xiangfeng

first: Wu

last: Nicholas

first: Deng

last: Xing Wang

first: Chen

last: Runsheng

first: Li

last: Lei


title: Functional genomic profiling of Aspergillus fumigatus biofilm reveals enhanced production of the mycotoxin gliotoxin

city: Jena

first: Daniela

last: Albrecht

address: Wildenbruchstr. 15

country: Germany

institute: BioControl Jena GmbH

summary: The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and, in part, immunocompetent patients. Like bacteria or yeast, A. fumigatus can grow in multicellular communities by the formation of a hyphal network encased in an extracellular matrix. Here, we describe the proteome and transcriptome of planktonic and biofilm-grown A. fumigatus mycelium after 24h and 48h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes encoding hydrophobins and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in biofilm cultures. This was confirmed by RT-PCR and by detection of this immunologically active mycotoxin in culture supernatants using HPLC analysis. The enhanced production of gliotoxin by in vitro formed biofilms reported here may play also a significant role under in vivo conditions. It may confer A. fumigatus protection from the host immune system and also enable its survival and persistence in chronic lung infections such as aspergilloma.

overall_design: Comparison of biofilm and submers cultures at 24h and 48h after induction.

first: Sandra

last: Bruns

first: Marc

last: Seidler


title: Meta-transcriptome analysis of a natural spelt sourdough ecosystem during a 10-day spontaneous laboratory fermentation

status: Public on Jul 07 2010

city: Leuven

first: Rekin's

last: Janky

address: Herestraat 49 Box 816

department: Nucleomics Core

country: Belgium

institute: VIB

summary: Lactic acid bacteria (LAB) are of industrial importance in the production of fermented foods, among which sourdough-derived products. Despite their limited metabolic capacity LAB contribute considerably to important characteristics of fermented foods, among which extended shelf-life, microbial safety, improved texture, and enhanced organoleptic properties. Thanks to the considerable amount of LAB genomic information that became available during the last years, transcriptome, and by extension meta-transcriptome studies, are the exquisite research approaches to study whole ecosystem gene expression into more detail.  In this study, microarray analyses were performed using RNA sampled during four 10-day spontaneous sourdough fermentations carried out in the laboratory, namely two wheat and two spelt fermentations with daily back-slopping. Hereto, the in-house developed functional gene LAB microarray was used, representing 406 genes that play a key role in sugar and nitrogen metabolism, functional metabolite production, stress responses and health and safety characteristics. The results reveal the activation of different key metabolic pathways, the ability to use different energy sources, and successful acid and oxidative stress responses. Also, a new algorithm was developed to compute a net expression profile for each of the represented genes, thereby exceeding the species level.

sample_organism:
Leuconostoc mesenteroides
Oenococcus oeni
Pediococcus acidilactici
Pediococcus pentosaceus
Staphylococcus aureus
Streptococcus thermophilus
Enterococcus faecalis
Enterococcus faecium
Enterococcus hirae
Lactococcus lactis
Lactobacillus acidophilus
Levilactobacillus brevis
Lentilactobacillus buchneri
Lacticaseibacillus casei
Weissella confusa
Lactobacillus delbrueckii
Lactobacillus helveticus
Lentilactobacillus hilgardii
Lactiplantibacillus pentosus
Lactiplantibacillus plantarum
Lactobacillus gasseri
Lacticaseibacillus paracasei
Limosilactobacillus reuteri
Latilactobacillus sakei
Companilactobacillus alimentarius
Lactobacillus amylovorus
Companilactobacillus farciminis
Limosilactobacillus fermentum
Fructilactobacillus fructivorans
Fructilactobacillus sanfranciscensis
Bifidobacterium
Brevibacterium linens
Latilactobacillus curvatus
Lactobacillus johnsonii
Leuconostoc citreum
Leuconostoc pseudomesenteroides
Limosilactobacillus pontis
Enterococcus casseliflavus
Mammaliicoccus lentus
Limosilactobacillus panis
Lacticaseibacillus rhamnosus
Lactobacillus crispatus
Enterococcus mundtii
Lactiplantibacillus paraplantarum
Companilactobacillus paralimentarius
Limosilactobacillus frumenti
Lentilactobacillus parabuchneri
Companilactobacillus mindensis
Furfurilactobacillus rossiae

overall_design: The labeled aRNA of the sourdough fermentation samples was hybridized using a loop design, i.e. subsequent samples (e.g. 27 h and 51 h, 51 h and 75 h etc.) were hybridized together on the microarray and the loop was closed by hybridizing the last sample with the first.

first: Joke

last: Allemeersch

first: Stefan

last: Weckx

first: Roel

last: Van der Meulen

first: Gino

last: Vrancken

first: Luc

last: De Vuyst

first: Paul

last: Van Hummelen


title: Conditional Dmrt1 knockout gene expression (phase 7)

status: Public on Jun 30 2010

city: Minneapolis

first: Aaron

last: Sarver

state: MN

address: 425 East River Road

department: Masonic Cancer Center

institute: University of Minnesota

summary: Genome wide analysis of DNA binding and transcriptional regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis.

overall_design: Gene expression differences found in conditionally targeted P9 testes were compared to those of controls


title: CGH profiles of sporadic breast tumors

status: Public on Jul 05 2010

city: Hamburg

first: Simon

middle: A

last: Joosse

address: Martinistrasse 52

department: Institute of Tumor Biology

institute: University Medical Center Hamburg-Eppendorf

type: Genome variation profiling by genome tiling array

summary:
Genomic DNA from sporadic breast tumours was isolated and analysed using array CGH. The NKI 1MB BAC/PAC micro array was used to identify chromosomal aberrations of all tumours.
Keywords: sporadic breast tumour, CGH

overall_design: Locate chromosomal aberrations in sporadic breast tumours

first: Petra

middle: M

last: Nederlof


title: Global transcriptional response to carbonic anhydrase IX deficiency in the mouse stomach

city: Tampere

first: Heini

last: Kallio

address: Biokatu 6

department: Institute of Medical Technology and School of Medicine

country: Finland

institute: University of Tampere

summary:
Background
Carbonic anhydrases (CAs) are a family of enzymes that regulate pH homeostasis in various tissues. CA IX is an exceptional member of this family because in addition to the basic CA function, it has been implicated in several other physiological and pathological processes. Functions suggested for CA IX include roles in cell adhesion and malignant cell invasion. In addition, CA IX likely regulates cell proliferation and differentiation, which was demonstrated in Car9‾/‾ mice. These mice had gastric pit cell hyperplasia and depletion of chief cells; however, the specific molecular mechanisms behind the observed phenotypes remain unknown. Therefore, we wanted to study the effect of CA IX deficiency on whole-genome gene expression in gastric mucosa. This was done using Illumina Sentrix Mouse-6 Expression BeadChip arrays. The expression of several genes with notable fold-change values was confirmed by QRT-PCR.

Results
CA IX deficiency caused the induction of 86 genes and repression of 46 genes in the gastric mucosa. There was 92.9% concordance between the results obtained by microarray analysis and QRT-PCR. The differentially expressed genes included those involved in developmental processes and cell differentiation. In addition, CA IX deficiency altered the expression of genes responsible for immune responses and downregulated the expression of several digestive enzymes.

Conclusions
Microarray analysis identified several potential genes whose altered expression could explain the disturbed cell lineage phenotype in the Car9‾/‾ gastric mucosa. The results also indicated a novel role for CA IX in the regulation of immunologic processes and digestion. These findings reinforce the concept that the main role of CA IX is not the regulation of pH in the stomach mucosa. Instead, it is needed for proper function of several physiological processes.

overall_design: C57BL/6 mice were used for the study: 6 Car9 knockout mice (3 males and 3 females) and 6 wildtype mice as their controls (3 males and 3 females). Stomach tissues were analyzed from all these mice.

first: Mika

last: Hilvo

first: Alejandra

last: Rodriguez

first: Eeva-Helena

last: Lappalainen

first: Anna-Maria

first: Seppo

last: Parkkila


title: Composition of Staufen2 ribonucleoprotein particles in neurons

status: Public on Jul 06 2010

city: Montréal

last: DesGroseillers

state: Québec

address: 2900 Edouard Montpetit

department: Biochimie

country: Canada

institute: Université de Montréal

summary: In neurons, many mRNAs are transported along neuronal dendrites to their site of translation by ribonucleoprotein complexes (mRNPs). Numerous mRNA-binding, motor and regulatory proteins within mRNPs finely regulate the fate of each of these mRNAs and their specific combination is likely to define different types of mRNA complexes and their neuronal functions. Staufen2 is a well known mRNA-binding protein present in distinct population of mRNPs in neurons and is implicated in the transport of mRNAs along the dendritic microtubules. However, little is known about the associated mRNAs in Staufen2 neuronal mRNPs complexes. As a means to understand its role in neuronal processes, a genome wide approach has been used to identify mRNAs that are co-immunoprecipitated with Staufen2 complexes from embryonic rat brains. The genome wide approach identified 1780 mRNAs associated with Staufen2 mRNPs that code for proteins involved in cellular processes such as post-translational protein modifications, RNA metabolism, intracellular transport and translation. All these mRNAs are consistent with the role of Staufen2 in synaptogenesis.

overall_design: Immunoprecipitation of Staufen2-containing mRNPs was performed on cell extracts prepared from whole brains of E17-E18 Sprague Dawley rat embryos. Cells were dissociated with a manual putter and lysed in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Triton X-100, 15 mM EGTA, 1 mM DTT and complete EDTA-free protease inhibitor cocktail (Roche). Cell lysates were centrifuged at 10 000 rpm for 10 min. After centrifugation, the supernatants were incubated with a rabbit polyclonal anti-Staufen2 or preimmune antibodies for 2 hours at 4°C, then with a 50% protein A-sepharose slurry for 2 hours at 4°C. Immune complexes were washed five times with the lysis buffer and eluted from the resin by heating at 95°C for 5 minutes in elution buffer (100 mM Tris-HCl pH 7.4, 200 mM DTT and 4% SDS). RNAs were isolated by Trizol (Invitrogen) extraction. Experiments were repeated several times (Anti-Stau2: n = 6; control preimmune: n = 6).

first: Maher-Laporte

last: Marjolaine

first: DesGroseillers

last: Luc


title: Conditional Dmrt1 knockout gene expression (phase 8)


title: High Density custom Agilent 44K CGH array analysis of 7q and TET2 region in myelodysplastic/myeloproliferative neoplasms

city: Salisbury

first: Andy

last: Chase

address: Salisbury District Hospital

country: United Kingdom

institute: Wessex Regional Genetics Laboratory

summary: Abnormalities of chromosome 7q are common in myeloid malignancies. Agilent CGH arrays targeted to 7q and Affymetrix SNP 6.0 arrays were used to characterise 7q aUPD and deletions. Sample #017 in this data series showed a microdeletion encompassing EZH2. Screening of a total of 614 cases with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals, most commonly myelodysplastic/myeloproliferative neoplasms (27/219; 12%) and myelofibrosis (4/30; 13%). EZH2 encodes the catalytic subunit of the Polycomb repressive complex 2 (PRC2), the highly conserved histone H3 lysine 27 methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity and its overexpression has been causally linked to differentiation blocks in epithelial tumors. Unexpectedly, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies.

overall_design: High density CGH array analysis of 7q and the TET2 region in 8 atypical myeloproliferative neoplasms

first: Andrew

first: Thomas

last: Ernst

first: Nicholas

middle: P

last: Cross


title: Conditional Dmrt1 knockout gene expression (phase 6)


title: Genome expression between homozygous and heterozygous yeast wine strains

city: Santiago

first: Ulises

last: Urzua

address: Independencia 1027

department: Basic and Clinical Oncology

country: Chile

institute: University of Chile

summary: Genome expression analysis between the yeast wine strain L-846 (diploid heterozygous) and spore derived from it (diploid homozygous).

overall_design: Slides contained cDNA clones spotted in duplicate. A repeated dye-swap design was used.

first: Francisco

last: Salinas


title: A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

city: Orsay

first: Frederique

last: Bidard

address: Bâtiment 400

department: SIGNAL TRANSDUCTION, REPRODUCTION & DEGENERATIVE PROCESSES

country: France

institute: Institut de Génétique et Microbiologie UMR 8621

type:
Expression profiling by array
Genome variation profiling by array

summary:
A genome wide microarray, covering the 10546 presently known and predicted CDS, has been constructed with the Agilent in-situ synthesized 60-mers technology.
1) A library of 10 oligonucleotides per CDS has been design by the manufacturer.
2) A computational step has been performed to determine 3' position, intron position and cross-hybridization rate of all probes. These data have been compiled to attribute a score to each probe and thus, to select a first set of 4 oligonucleotides per CDS.
3) Microarray v.2 (GPL10115), manufactured with this design, have been used for a selection procedure based on the use of transcriptomic experimental data generated with diverse culture conditions. To maximize the number of expressed CDS, experimental data were generated with a large range of growth conditions covering crucial physiological changes from key steps of vegetative growth and sexual development.For each condition, four biological replicates were done to increase the statistical power of the analysis. An oligonucleotide scoring has been settled to select the best oligonucleotide per CDS.

The final array design, Microarray v.3 (GPL10116), contained 10,539 probes for nuclear CDS. The array was enriched with 17 mitochondrial CDS probes that did not undergo the experimental screening. Each probe was replicated four times on each 44K array to improve the statistical significance of results. Before using in industrious transcriptomic experiment, ultimate qualification steps have been carried out. First, DNA extracted from the/ S/ and /s /strains was hybridized with the final design Microarray v.III. The apha_ORF and /het/ genes were identified as polymorphic between /s/ and /S/ strains (|FC|>2; a/ =/0.001) confirming previous genetic and molecular analyses (Turcq B etal.,1990*,* /Current genetics, /Sellem et al., 1990, Mol. Gen. Genet.). In second qualification step, a self-to-self hybridization experiment, replicated three times, has been /per/formed with a commonreference: 89.3% of the probes displayed a signal-to-standard-deviation ratio >3. The final microarray design is referenced as AMADID 018343. Microarray slides are available to the research community from Agilent.

overall_design:
We developed oligonucleotide microarrays for the filamentous fungus Podospora anserina as part of the ANR program project grant ANR-05-BLAN-0385 (project SexDevMycol), coordinated by R. Debuchy. This project was aimed to analyze the sexual development of /P. anserina/ with a variety of approaches, including transcriptomic profiling. Different versions of microarrays were tested on mycelium and sexual development time courses, to evaluate their perfomance in comparing transcriptomic profiles of vegetative vs sexual stages, and in analyzing transcriptomic profiles evolution during sexual development. Complete analyses were performed with the microarray final version (v.III) and several manuscripts dealing with these topics are in preparation.
The s (small s) and S (big S) strains of Podospora anserina are different geographical isolates of the same species. Both strains can cross and have identical ITS (accession #: AY278557 and GU391422) confirming that they belong to the same species. The S strain has been completely sequenced (Espagne et al., Genome Biology, 2008, 9, R77 ) and its genome is currently at the finishing step (P. Silar and P. Wincker, unpublished results). The genome of the s strain is not sequenced, and only a few genes are known. The major known difference is at the level of the s locus controling vegetative incompatibility (Turcq et al., Current genetics 1990, 17:297-303).

PART1 (with GPL10115): reference design, 5 conditions each with four biological replicates : M24h, M48h, M96h, C24h, C48h; common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5

PART2 (with GPL10116): DNA hybridization with a direct design, wt s versus wt S, dye swap (four technical replicates);  RNA Homotypic hybridization with the common reference sample (three technical replicates)

first: Sandrine

last: Imbeaud

first: Nancie

last: Reymond

first: Robert

last: Debuchy

first: Hervé

last: Delacroix

first: Olivier

last: Lespinet

first: Véronique

last: Berteaux


title: Mhyo IG Rnd vs MhRnd

city: Ames

first: Chris

last: Minion

state: IA

address: 1130 Vet Med

department: VMPM

institute: Iowa State University

summary: Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist. The virulence factors of M. hyopneumoniae are largely unknown and are most probably complex in nature. The transcriptional profile of intergenic regions is investigated using microarray PCR and oligonucleotide probes. It is hypothesized that intergenic regions of the Mhyo genome are being transcribed along with the ORFs.

overall_design: To test for transcripts in IG regions, cDNA was generated from M. hyopneumoniae RNA using random hexamers (IDT; Integrated DNA Technologies, Inc. Coralville, IA)  and a set of 129 hexamer oligonucleotide primers designed for M. hyopneumoniae mRNA. A single total RNA sample was split into eight 5 μg samples, and cDNA synthesis was conducted on four of these samples using the random hexamers (IDT); for the other four samples, cDNA was generated using primers designed for M. hyopneumoniae as described by Madsen et al. 2006. In each set of four, two samples were fluorescently labeled with Cy3 and the other two were labeled with Cy5 using an indirect labeling protocol. After labeling and cleanup, all 8 samples were quantified on a Nanodrop ND-1000 spectrophotometer. For each array on the slide, 1 μg of labeled cDNA from each type of primer generation (Cy3 or Cy5, random or specific primer set) was combined and hybridized to a PCR featured array and an oligonucleotide featured array. This resulted in 2 arrays of PCR probes hybridized with dye swaps and 2 arrays of oligonucleotide probes with dye swaps. A second RNA sample was split in four 5 μg samples of total RNA, and the hybridization process was repeated for the oligonucleotide probe array.

first: Stuart

middle: W

last: Gardner


title: Study of acquired thermotolerance response in peanut using oligonucleotide microarrays

city: Lubbock

first: Kameswara Rao

last: Kottapalli

state: TX

address: 3810 4th Street

department: Plant & Soil Science

institute: USDA-ARS Lubbock & Texas Tech University

summary: To gain insights into molecular mechanisms of tolerance to heat stress, we conducted a transcript profiling experiment to identify heat-responsive genes in contrasting peanut mini core accessions, either un-acclimated or acclimated to heat stress.  Plants at reproductive stage were exposed to 28 °C (control), 45 °C for 15 d (un-acclimated) or 45 °C for 1 d followed by 7 d recovery and 15 d stress (acclimated).  Two contrasting genotypes showing diverse response to stress were selected based on a bioassay involving chlorophyll fluorescence yield under elevated respiratory demand and membrane thermostability.  Transcript profiling was performed using 8 x 15k custom oligo microarrays containing 15k peanut EST sequences.  Gene enrichment analysis was performed using Blast2GO program and genes with homology to known proteins were categorized into detailed molecular functional groups.  Majority of stress-responsive genes assigned to KEGG pathways belonged to starch, sucrose and galactose metabolism followed by aminoacid metabolism, and secondary metabolite biosynthesis.  Differentially expressed transcripts from samples were validated in the samples from second year by quantitative real-time PCR.  Transcripts of eight genes involved in terpenoid and flavanoid biosynthesis were induced after second and seventh day, respectively in leaves under heat stress.  Metabolite analysis confirmed increases in metabolites of selected pathways under heat stress.  The heat up-regulated genes in tolerant COC041 mini-core accession are potential candidate genes for engineering stress-tolerant peanuts and unraveling molecular mechanisms of peanut adaptation to heat stress.

overall_design: We used Agilent peanut microarrays to identify putative heat stress-responsive genes. Acclimated leaf tissues of the peanut genotypes COC041 (tolerant) and COC166 (susceptible) were used in the study. Three replications of microarray experiments were carried out by hybridizing the cRNA from different time points and stress conditions in a loop design on 8 x 15k microarray.

first: KameswaraRao

first: Paxton

last: Payton


title: Expression analysis of trichostatin A-treated Aspergillus fumigatus Af293

city: Wako

first: Takayuki

last: Motoyama

state: Saitama

address: 2-1 Hirosawa

country: Japan

institute: RIKEN

summary: Investigation of whole genome gene expression level changes in trichostatin A (TSA)-treated A. fumigatus Af293 compared to non-treated A. fumigatus Af293.

overall_design: Species: Aspergillus fumigatus; Strain: Af293; Type of array: Eukaryotic Expression (4plex, 2plex for a TSA-treated sample and 2plex for a non-treated sample); Technical replicates: Two per treatment.

first: Hiroyuki

last: Osada


title: Paralogous MarR/DUF24-family repressors YodB and CatR control expression of the catechol dioxygenase CatE in Bacillus subtilis

status: Public on Jun 29 2010

city: Greifswald

first: Haike

last: Antelmann

address: F.-L.-Jahnstr. 15

department: Institute for Microbiology

institute: University of Greifswald

summary: The redox-sensing MarR/DUF24-type repressor YodB controls expression of the azoreductase AzoR1 and the nitroreductase YodC that are involved in detoxification of quinones and diamide in Bacillus subtilis. In the present paper, we identified YodB and its paralog YvaP (CatR) as repressors of the yfiDE (catDE) operon encoding a catechol-2,3-dioxygenase that also contributes to quinone resistance. Inactivation of both paralogs, CatR and YodB, results in full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis studies showed that CatR protects two inverted repeats with the consensus sequence TTAC-N5-GTAA overlapping the -35 promoter region (BS1) and the transcriptional start site (BS2). The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue that is essential for redox-sensing of CatR in vivo as shown by Cys-to-Ser mutagenesis. Our data suggest that CatR is modified by intermolecular disulfide formation in response to diamide and quinones in vitro and in vivo. Redox-regulation of CatR occurs independently of YodB and no protein interaction was detected between CatR and YodB in vivo using protein-crosslinking and mass spectrometry.

overall_design: Bacillus subtilis 168 wild type (Ko_WT) control vs. DyvaP mutant  (Ko_DyvaP). The experiment was conducted in duplicates using two independent total RNA preparations. For all datasets used for final analysis, wild-type samples were labeled with Cy3 and DyvaP mutant samples were labeled with Cy5.

first: Bui

last: Chi Khanh

first: Kazuo

last: Kobayashi

first: Dirk

last: Albrecht

first: Michael

last: Hecker


title: Mushroom development in S. commune

city: Utrecht

last: Wösten

address: Padualaan 8

country: Netherlands

institute: Utrecht University

summary: In order to study changes in gene expression during mushroom development in Schizophyllum commune, genome wide gene expression was analysed in 4 developmental stages: vegetative mycelium, stage I aggregates, stage II primordia and mature mushrooms

overall_design:
4 samples:
- vegetative mycelium
- stage I aggregates
- stage II primordia
- mature mushrooms
RNA was obtained from 3 biological replicates and pooled

last: Ohm

last: De Jong

last: Lugones


title: Expression profiling of GIST: CD133 is associated with KIT exon 11 deletion, gastric location and poor prognosis

city: Göteborg

first: Erik

last: Kristiansson

address: Chalmers Tvärgata 3

department: Department of Mathematical Sciences

country: Sweden

institute: Chalmers University of Technology

summary:
Background & Aims: In gastrointestinal stromal tumors (GIST) KIT exon 11 deletions are associated with poor prognosis. The aim of this study was to determine the gene expression profile of GIST carrying KIT exon 11 deletions and to identify genes associated with poor prognosis.
Methods: Expression profiling was performed on 9 tumors with KIT exon 11 deletions and 7 without KIT exon 11 mutations using oligonucleotide microarrays. In addition, gene expression profiles for 35 GISTs were analysed by meta-analysis. Differentially expressed genes were identified and confirmed by qPCR. Expression of CD133 (prominin-1) protein (AC133) was also examined by tissue microarray (TMA) analysis of 204 GISTs from a population-based study in Western Sweden. Survival analysis was performed using Cox regression model.
Results: Gene expression profiling, meta-analysis and qPCR demonstrated up-regulation of the stem cell marker CD133 in GIST carrying KIT exon 11 deletions. Immunohistochemical analysis on TMA confirmed CD133 expression in 28% of all tumors. CD133 positivity was frequent in gastric GIST (48%) versus small intestinal GIST (4%). CD133 positivity was also frequent in GIST with KIT exon 11 mutations (41%) compared to tumors with mutations in KIT exon 9, PDGFRA, or wild-type tumors (0-17%). There was no significant correlation between CD133 staining and NIH risk score. Survival analysis demonstrated significant correlation between presence of CD133 and shorter overall survival.
Conclusions: The stem cell marker CD133 is expressed in a subset of predominantly gastric GIST with KIT exon 11 mutations and associated with poor prognosis.

overall_design: Tumors with and without KIT exon 11 deletion were compared using a common reference design.

first: Gabriella

last: Arne

first: Olle

last: Nerman

first: Lars-Gunnar

last: Kindblom

first: Håkan

last: Ahlman

first: Bengt

last: Nilsson

first: Ola


title: Tiled Array Experiments Determining RNA Expression in gHV68 infected 3T12 cells

status: Public on Jun 28 2010

city: St. Louis

first: Steven

last: Johnson

state: MO

address: 660 S. Euclid

department: Department of Pathology and Immunology

institute: Washington University-School of Medicine

type: Expression profiling by genome tiling array

type: Non-coding RNA profiling by genome tiling array

summary: Viral genetic studies often focus on large open reading frames (ORFs) identified during genome annotation (ORF-based annotation).  Here we provide a tool and software set for defining gene expression by murine gammaherpesvirus 68 (γHV68) nucleotide-by-nucleotide across the 119,450 basepair (bp) genome.  These tools allowed us to determine that viral RNA expression was significantly more complex than predicted from ORF-based annotation, including over 73,000 nucleotides of unexpected transcription within 30 expressed genomic regions (EGRs).  Approximately 90% of this RNA expression was antisense to genomic regions containing known large ORFs.  We verified the existence of novel transcripts in three EGRs using standard methods to validate the approach and determined which parts of the transcriptome depend on protein or viral DNA synthesis.  This redefines the genetic map of γHV68, indicates that herpesviruses contain significantly more genetic complexity than predicted from ORF-based genome annotations, and provides new tools and approaches for viral genetic studies.

overall_design: Study includes a total of 12 samples. These are 3 replicates of gHV68-infected 3T12 cells 18hpi, 3 replicates of gHV68-infected 3T12 cells 8hpi, 3 replicates of gHV68-infected 3T12 cells 8hpi treated with cyclohexamide, and 3 replicates of gHV68-infected 3T12 cells 18hpi treated with cidofovir. Each of these samples has a matching mock-infected 3T12 sample which was subtracted to remove host RNA crosshybridization and are attached as extra raw data files.

first: L

middle: S

first: Erin

middle: K

last: Willert

first: Herbert

last: Virgin


title: The activation potential of MOF is constrained for dosage compensation

status: Public on Jun 26 2010

city: Munich

first: Christian

last: Feller

address: Schillerstraße 44

department: Molecular Biology

institute: Adolf-Butenandt Institute at the LMU University Munich

type: Genome binding/occupancy profiling by genome tiling array

summary:
The H4K16 acetyltransferase MOF plays a crucial role in dosage compensation in Drosophila, but has additional, global functions. We compared the molecular context and effect of MOF in male and female flies combining chromosome-wide mapping and transcriptome studies with analyses of defined reporter loci in transgenic flies. MOF distributes dynamically between two complexes, the Dosage Compensation Complex and a complex containing MBD-R2, a global facilitator of transcription. These different targeting principles define the distribution of MOF between the X chromosome and autosomes and at transcription units with 5’ or 3’ enrichment.
The male X chromosome differs from all other chromosomes in that H4K16 acetylation levels do not correlate with transcription output. The reconstitution of this phenomenon at a model locus revealed that the activation potential of MOF is constraint in male cells in the context of the DCC to arrive at the two-fold activation of transcription characteristic of dosage compensation.

overall_design: ChIP-chip profiling of MBD-R2, MOF and MSL1 in adult male and female flies, and SL2 cells, incl. at least 3 biological replicates

first: Peter

last: Becker


title: Comparison of endogenous and viral microRNA profile of Arabidopsis thaliana with and without Turnip Mosaic Virus infection.

status: Public on Jun 27 2010

city: Brisbane

first: Shazia

last: Iram

address: John Hines building # 62

department: School of Biological Sciences

country: Australia

institute: University of Queensland

summary: Individual miRNA profile was identified for two Col-0 WT replicates and two Col-0 TuMV infected plant replicate samples, with 12-15 plants in each replicate.Comparison was done based on the microRNA expression profile in both biological replicates for infected and uninfected plant samples .

overall_design: 12-15 plants were used in all sample replicates independently.

first: Peer

last: Schenk


title: Expression profiling of heat stress response in peanut using oligonucleotide microarrays

first: Kameswara Rao

last: Kottapalli

summary: To gain insights into molecular mechanisms of tolerance to heat stress, we conducted a transcript profiling experiment to identify heat-responsive genes in contrasting peanut mini core accessions, either un-acclimated or acclimated to heat stress.  Plants at reproductive stage were exposed to 28 °C (control), 45 °C for 15 d (un-acclimated) or 45 °C for 1 d followed by 7 d recovery and 15 d stress (acclimated).  Two contrasting genotypes showing diverse response to stress were selected based on a bioassay involving chlorophyll fluorescence yield under elevated respiratory demand and membrane thermostability.  Transcript profiling was performed using 4 x 44k custom oligo microarrays containing 22k peanut EST sequences.  The microarray analysis identified 710 stress-induced and 770 stress-repressed putative heat-responsive transcripts in the tolerant genotype.  Gene enrichment analysis was performed using Blast2GO program and genes with homology to known proteins were categorized into detailed molecular functional groups.  Majority of stress-responsive genes assigned to KEGG pathways belonged to starch, sucrose and galactose metabolism followed by amino acid metabolism, and secondary metabolite biosynthesis.  Differentially expressed transcripts from samples obtained from first year’s experiment were validated in the samples from second year by quantitative real-time PCR.  Transcripts of eight genes involved in terpenoid and flavanoid biosynthesis were induced after second and seventh day, respectively, in leaves under heat stress.  Metabolite analysis confirmed increases in metabolites of selected pathways under heat stress.  The heat up-regulated genes in tolerant COC041 mini-core accession are potential candidate genes for engineering stress-tolerant peanuts and unraveling molecular mechanisms of peanut adaptation to heat stress.

overall_design: We used Agilent peanut microarrays to identify putative heat stress-responsive genes. Directly heat-stressed leaf tissues of the peanut genotypes COC041 (tolerant) and COC166 (susceptible) were used in the study. Three replications of microarray experiments were carried out by hybridizing the cRNA from different time points and stress conditions in a loop design on 4 x 44k microarray.

first: KameswaraRao

first: Paxton

last: Payton


title: Molecular influences relating to the development and biomineralization of the chiton radula, Acanthopleura histosa

status: Public on Jun 25 2010

city: Pacific Grove

first: Luke

last: Gardner

state: California

address: 120 Ocean View Blvd, Hopkins Marine Station

institute: Stanford University

summary: Chitons are marine molluscs that posses a radula, a tongue like appendage containing many rows of microscopic teeth used for feeding. These teeth are hardened through the incorporation of biominerals. Since the original discovery of iron oxide and magnetite in these teeth, a wealth of chemical and structural knowledge has accumulated. The biomineralization process is known to be matrix mediated with precise control of the deposition of a number of different iron and calcium minerals at readily identifiable stages of tooth development. While much is known about the mechanisms of tooth mineralisation, there have been no studies undertaken to identify the genes that regulate this process. This investigation uses microarray technology to analyse the spatial expression of 493 expressed sequence tags (EST) derived from the radula sac of chiton, Acanthopleura hirtosa. A number of ESTs have been deemed significantly differentially expressed in relation to three designated areas of the radula sac. These spatial sections are defined by the variation in biomineral deposits of the radula, being immature radula which lacks any biomineral incorporation, iron deposition and calcium deposition. Additionally, foot muscle was used as a control to specify whether EST expression was specific to the radula and therefore likely involved biomineralization.

overall_design: A total of twelve A. hirtosa specimens were collected and sacrificed for microarray hybridizations. The radulae were removed from each animal immediately following sacrifice and dissected into three sections based on the biomineralisation development of the radula teeth. These sections are termed immature teeth, iron deposited teeth, and calcium deposited teeth. An additional sample of A. hirtosa foot muscle was taken from each specimen. RNA was extracted individually from these sections. Extracted RNA samples across subjects were pooled equally within sections in order to minimize the impact of biological variation and maximize the number of individual samples included in the study. To this effect, four single channel hybridizations were conducted for each radula and muscle section each representing a pooled biological replicate containing equal proportions of RNA from three A. hirtosa individuals. A total of sixteen microarray hybridizations were conducted in this manner.

middle: D

first: Lesley

last: Brooker

first: Jeremy

last: Shaw

first: Abigail

last: Elizur


title: Expression profiling in the larval chemosensory organ of different prospero mutants

city: Nantes

first: Mahatsangy

last: Raharijaona

address: Faculte de Medecine - Inserm U915 1 rue gaston Veil

institute: INSERM

summary:
The antenno-maxilary complex (AMC) forms the chemosensory system of the Drosophila larva and is involved in gustatory and olfactory perception. We have previously shown that a mutant allele of the homeodomain transcription factor Prospero (prosVoila1, V1), presents several developmental defects including abnormal growth and altered taste responses. In addition, many neural tracts connecting the AMC to the central nervous system (CNS) were affected. Our earlier reports on larval AMC did not argue in favour of a role of pros in cell fate decision, but strongly suggested that pros could be involved in the control of other aspect of neuronal development. In order to identify these functions, we used microarray analysis of larval AMC and CNS tissue isolated from the wild type, and three other previously characterised prospero alleles, including the V1 mutant, considered as a null allele for the AMC.
A total of 17 samples were first analysed with hierarchical clustering. To determine those genes affected by loss of pros function, we calculated a discriminating score reflecting the differential expression between V1 mutant and other pros alleles. We identified a total of 64 genes in the AMC. Additional manual annotation using all the computed information on the attributed role of these genes in the Drosophila larvae nervous system, enabled us to identify a first functional category of potential Prospero target genes known to be involved in neurite outgrowth, synaptic transmission and more specifically in neuronal connectivity remodelling. The second category of genes found to be differentially expressed between the null mutant AMC and the other alleles concerned the development of the sensory organs and more particularly the larval olfactory system. Surprisingly, a third category emerged from our analyses and suggests an association of pros with the genes that regulate autophagy, growth and insulin pathways. Interestingly, EGFR and Notch pathways were represented in all of these three functional categories. We now propose that Pros could perform all of these different functions through the modulation of these two antagonistic and synergic pathways.
The current data contribute to the clarification of the Prospero function in the larval AMC and show that pros regulates different function in larvae as compared to those controlled by this gene in embryos. In the future, the possible mechanism by which Pros could achieve its function in the AMC and the possible involvement of EGFR and Notch pathway will be explored in detail

overall_design:
Gene expression was measured in two different tissues (Brain and AMC) of prospero mutants flies (V1, V13, V14, V24).
3 biological replicates were performed for V1, V13 and V24 AMC and V14 Brain;
2 biological replicates for V14 AMC and V13 Brain.

first: Laure

last: Guenin

first: Rémi

last: Houlgatte

first: Fawzia

last: Baba-Aissa


title: Ebf1 ChIP

status: Public on Jun 24 2010

city: Freiburg

first: Rudolf

last: Grosschedl

address: Stuebeweg 51

department: Cellular and Molecular Immunology

institute: Max Planck Institute of Immunobiology

summary: Ebf1 is a key determinant of B-lymphocyte specification. In order to identify unknown transcriptional targets, endogenous Ebf1 was isolated by chromatin-IP from primary pro-B cells and the copurified DNA was hybridized to promoter tiling arrays.

overall_design: Ebf1 was ChIPed using a polyclonal antibody directed against an N-terminal peptide from primary pro-B cells grown in the presence of Il7. The resulting DNA was analyzed by tiling array hybridization.

first: Treiber

last: Thomas

first: Firner

last: Sonja

first: Grosschedl

last: Rudolf


title: Expression profiling of over-expression of conspecific and heterspecific miR-310 cluster in Drosophila (cDNA array)

city: Guangzhou

first: TIAN

last: TANG

address: Xingangxi Rd. #135

country: China

institute: Sun Yat-Sen University

summary: The miRNA 310-311-312-313 cluster from D. melanogaster (referred to as Dm310s) and the homolog from D. pseudoobscura (Dp310s) were used to transform D. melanogaster. The over-expression of UAS-miR310s in the Dm310s transgenic line M1-7 and Dp310s transgenic line P4-18 was driven by GAL4 line NP5941 with native miR-310 expression pattern. We used DGRC-1 cDNA arrays to assess the effects of miR310s over-expression on the whole transcriptome.

overall_design: Direct two-colour design involving six arrays in three dye-swap pairs, with each pair used for the pairwise comparison of Dm310s, Dp310s and w1118 (control).

first: Supriya

last: Kumar

first: Tian

last: Tang


title: Location-specific responses to thermal stress in larvae of the reef-building coral Montastraea faveolata

status: Public on Jun 23 2010

city: University Park

first: Nicholas

last: Polato

state: PA

address: 208 Mueller Lab

department: Biology

institute: Penn State University

summary: The potential to adapt to a changing climate depends in part upon the standing genetic variation present in wild populations. In corals, the dispersive larval phase is particularly vulnerable to the effects of environmental stress. Larval survival and response to stress during dispersal and settlement will play a key role in the persistence of coral populations. To test the hypothesis that larval transcription profiles reflect population specific responses to thermal stress, symbiont-free gametes of the scleractinian coral Montastraea faveolata were collected from Florida and Mexico and raised under normal and elevated temperatures. These populations have been shown to exchange larvae frequently enough to prevent significant differentiation of neutral loci. Differences among thousands of genes were simultaneously characterized using microarrays, allowing investigation of gene expression patterns among wild populations under stressful environmental conditions. Results show site-specific signatures of gene expression in larvae of a reef-building coral from different parts of its range (despite low genetic divergence), and reveal both local and general components of stress response during later stages of larval development. These results provide evidence of site-specific variation in the face of gene flow, which may represent functional genetic variation in different subpopulations, and support the idea that coral host genomes may indeed house the adaptive potential needed to deal with changing environmental conditions.

overall_design: The experimental setup followed a reference design, i.e. all samples were hybridized against the same pool made up of equal amounts of RNA from all samples collected in Mexico. For samples from Mexico we used three technical replicates for each treatment temperature, for samples from Florida three biological replicates were used for each treatment temperature, except for the high temperature samples at day two where only two replicates were available due to high larval mortality at that temperature. Common reference samples were labeled with Cy3, temperature treatment samples with Cy5. Microarrays for M. faveolata contained 1,314 coding sequences, of which 43% had functional annotations as determined by homology to known genes.

first: N

middle: R

first: C

last: Voolstra

first: J

last: Schnetzer

first: M

last: DeSalvo

middle: J

last: Randall

first: A

last: Szmant

last: Medina

first: I

middle: B

last: Baums


title: Genomic profiling of gastroesophageal adenocarcinoma

city: Lund

first: Anna

middle: Isinger

last: Ekstrand

address: Barngatan 2b

department: Oncology

institute: Clinical Sciences

summary: We aimed to characterize the genomic profiles of adenocarcinomas in the gastroesophageal junction in relation to cancers in the esophagus and the stomach. Profiles of gains/losses as well as gene expression profiles were obtained from 27 gastroesophageal adenocarcinomas using 32k high-resolution array-based comparative genomic hybridization (aCGH) and 27k oligo gene expression arrays and putative target genes were validated in an extended series. Adenocarcinomas in the distal esophagus and the gastroesophageal junction showed strong similarities with the most common gains at 20q13, 8q24, 1q21-q23, 5p15, 13q34, and 12q13, whereas different profiles with gains at 5p15, 7p22, 2q35, and 13q34 characterized gastric cancers.  CDK6 and EGFR were identified as putative target genes in cancers of the esophagus and the gastroesophageal junction with upregulation in one quarter of the tumors. Gains/losses and gene expression profiles show strong similarity between cancers in the distal esophagus and the gastroesophageal junction with frequent upregulation of CDK6 and EGFR, whereas gastric cancer displays distinct genetic changes. These data suggest that molecular diagnostics and targeted therapies can be applied to adenocarcinomas of the distal esophagus and gastroesophageal junction alike.

overall_design: Three types of gastroesophageal adenocarcinomas; 7 distal esophageal (EA) tumors, 9 junctional (JA) and 7 proximal stomach cancers (GA) was genomically profiled using  tiling 32k BAC-arrays. Two replicates of tumors IDs 1-EA-CGH (replicate 1-EA-CGH-2), 12-EA-CGH (replicate 12-EA-CGH-2) and 18-EA-CGH (replicate 18-EA-CGH-2).  Reference samples consist of a pool of male DNA (Promega, Madison, WI, USA).


title: Aortic arch profiling of Ldlr knockout mice with human CETP transgene

city: Seattle

first: Xia

last: Yang

state: WA

address: 1100 Fairview Ave N

institute: Sage Bionetworks

summary: Aortic arch profiling of Ldlr-/- huCETP transgenic mice fed a low fat, cholesterol-containing western diet (LFWD, 9% fat, 0.15% cholesterol) starting at 8 weeks of age for 4, 8, 12, and 16 weeks.

overall_design: Ldlr-/- huCETP transgenic mice were fed a low fat, cholesterol-containing western diet (LFWD, 9% fat, 0.15% cholesterol) starting at 8 weeks of age for 4, 8, 12, and 16 weeks (Supplementary Figure 1). At each time point, 10 mice were studied. Mice were euthanized at each time point and the aortic arch of each mouse was immediately collected and flash-frozen in liquid N2.   Mice were fasted overnight prior to euthanasia.  the aortic arches of 10 mice in the same study group were pooled for profiling, resulting in one pool per study group. Tissues were homogenized and total RNA was extracted. The control RNA pool was composed of equal aliquots of RNA derived from the 4 pools (40 samples) from the LFWD 4-, 8-, 12- and 16-week time points.


title: Nitrate Stress in Desulfovibrio vulgaris Hildenborough

status: Public on Jun 22 2010

city: Berkeley

first: Adam

middle: P.

last: Arkin

state: CA

address: 1 Cyclotron Road

department: Physical Biosciences Division

institute: Virtual Institute for Microbial Stress and Survival

summary:
Using data from microarray experiments, we investigated the effects of excess sodium nitrate on Desulfovibrio vulgaris.

Keywords: stress response, time course

sample_organism: Nitratidesulfovibrio vulgaris str. Hildenborough

overall_design: Comparison of cells treated with 105 mM sodium nitrate to untreated cells at times of 0, 30, 60, 120, and 240 min.


title: au08-06_mpk6_heat_stressed-Transcriptomic analysis of mpk6 KO mutant after heat stress treatment

city: evry

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

institute: INRA

summary:
au08-06_mpk6_heat_stressed - au08-06_mpk6_heat_stressed - Mitogen Activated Protein Kinase (MAPK) signaling pathways are key regulators of cell proliferation, differentiation and stress effectors. The core of the MAP kinase signal transduction cascade is composed of a three-kinase module consisting of a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK). The signaling pathway is activated upon stimulation by a phosphorylation cascade. In previous studies, it was shown that the mpk6 KO mutant plants are significantly more tolerant to heat stress in comparison to wt and that after 3h treatment at 37°C, an activation of heat-shock proteins occures in the mpk6 mutant. To better understand the changes occuring in the mpk6 mutant upon heat stress at the gene expression level, we would like to perform a microarray transcriptomic analysis. - Mitogen Activated Protein Kinase (MAPK) signaling pathways are key regulators of cell proliferation, differentiation and stress effectors. The core of the MAP kinase signal transduction cascade is composed of a three-kinase module consisting of a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK). The signaling pathway is activated upon stimulation by a phosphorylation cascade. In previous studies, it was shown that the mpk6 KO mutant plants are significantly more tolerant to heat stress in comparison to wt and that after 3h treatment at 37°C, an activation of heat-shock proteins occures in the mpk6 mutant. To better understand the changes occuring in the mpk6 mutant upon heat stress at the gene expression level, we would like to perform a microarray transcriptomic analysis.

Keywords: treated vs untreated comparison

overall_design: 4 dye-swap - CATMA arrays

first: Concetta

last: Giuliani

first: Heribert

last: Hirt

first: Jean-Pierre

last: Renou

first: Marie-Laure

last: Martin-Magniette


title: A quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

city: Toronto

first: Patricia

last: Reis

state: Ontario

address: 610 University Avenue

department: Applied Molecular Oncology

institute: University Health Network

summary:
MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p<0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples.  We outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30<Ct<35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum.  We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p<0.001), when examining all miRs, regardless of RNA extraction method used.  Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance).
Our study aims to demonstrate the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

overall_design: FFPE samples from 4 benign, reactive lymph nodes and 3 mantle cell lymphomas. Paired fresh-frozen and FFPE samples (3 benign reactive lymph nodes and 3 mantle cell lymphomas) were used for comparison. We also tested RNA samples at different concentrations (10, 25, 50, 100 and 200 ng/uL) as well as duplicate experiments for these RNA concentrations. Distinct RNA dilutions were also compared (5X, 7.5X, 30X and 62.5X dilution) at RNA concentrations of 66.7 ng/uL, 100 ng/uL and 200 ng/uL).

first: Rashmi

last: Goswami

first: Levi

last: Waldron

first: Jerry

last: Machado

first: Nilva

last: Cervigne

first: Wei

last: Xu

first: Denis

last: Bailey

first: Igor

last: Jurisica

first: Michael

last: Crump

first: Suzanne

last: Kamel-Reid


title: Structural Variation of the Mouse Natural Killer Gene Complex as Determined by High Resolution Array-based CGH Profiling

city: Saint Louis

first: Darryl

last: Higuchi

address: 660 S. Euclid Ave

department: Internal Medicine

institute: Washington University

summary: We used aCGH data for detailed analysis of the natural killer gene complex (NKC) of multiple inbred strains, providing insight both into aCGH analysis of polymorphic gene clusters as well as structural information on the NKC itself.  By unsupervised clustering analysis, the aCGH data can be precisely correlated with overall genome structural variation of the NKC as detected by RFLP variants in retrospective and prospective analyses.  On a high resolution level, aCGH could be used to detect a small deletion within an NKC gene that is confined to only one mouse strain examined out of 22.

overall_design: The NKC region on mouse chromosome 6 is known to be an area with structural variation based on studies which reveal restriction fragment length polymorphic (RFLP) variants among inbred mouse strains.  To verify predictive power of the unsupervised clustering algorithms, we prospectively collected aCGH data from two strains of mice (CBA/J, and NZW/LacJ) that were not among the original 20 strains (GEO accession # GSE10656) analyzed but represented strains whose Ly49 and Nkrp1 RFLP had been determined previously.  We performed comparative genomic hybridization using a long-oligonucleotide array containing 2,149,887 probes evenly spaced across the reference genome with a median inter-probe spacing of 1,015 bases.  Labeling, hybridization, washing, and array imaging were performed as previously described (PMID:18334530).  To capture the variability along chromosome 6, the log2 signal ratios were organized into bins (a defined chromosome length in terms of bp).  The entire chromosome was divided into consecutive bins, beginning with the position of the first probe.  We used the variance of each bin to represent the variability along the chromosome.  We performed two types of unsupervised clustering analysis of the log2 ratios of signal intensities of each probe (normalized).  One is the non-hierarchical clustering using the partitioning among medoids (PAM) function as implemented in R's 'cluster' package.  The other is the agglomerative hierarchical clustering based on the Euclidean distance.  The average linkage method was used in clustering the strains as implemented in the hclust function in R's 'cluster' package.

first: Wayne

last: Yokoyama


city: Amherst

first: Ashley

middle: Edwin

last: Franks

state: MA

address: University of Massachusetts at Amherst, Dept. of Microbiology, Morrill IV North

department: Microbiology

institute: University of Massachusetts Amherst

summary:
Microtoming Coupled with Microarray Analysis to Evaluate Potential Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in Anode Biofilms

Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in A Current Producing Biofilm

Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. In order to further compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, mature anode biofilms were treated to stop turnover over of mRNA and then encased in resin which was sectioned into 100 nm shavings with a diamond knife and pooled into inner (0-20 µm from anode surface) and outer (30-60 µm) fractions. Whole genome DNA microarray analysis of RNA extracted from the shavings revealed that, at a 2-fold lower threshold, there were 146 genes that had significant (p<0.05), differences in transcript abundance between the inner and outer portions of the biofilm. Only 1 gene, GSU0093, a hypothetical ABC transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting that cells in the outer biofilm had lower metabolic rates. However, the differences in transcript abundance were relatively low (<3-fold) and the outer biofilm did not have significantly lower expression of the genes for TCA cycle enzymes which previous studies have demonstrated are sensitive indicators of changes in rates of metabolism in G. sulfurreducens.  There also was no significant difference in the transcript levels for outer-surface cell components thought to be important in electron transfer in anode biofilms.  Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode.  The results of the metabolic staining and gene expression studies suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production.  The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms.

overall_design: Three biological replicates were hybridized in triplicate on a coustom affimetrix tilling array using prokaryotic protocol (p69Affy, p75 Adobe) for labeling, hybridization and scanning.

middle: E

first: Kelly

last: Nevin

first: Richard

middle: H

last: Glaven

first: Derek

last: Lovley


title: Measuring transcriptional differences between Caulobacter crescentus cultured in nitrate versus ammonium as the sole nitrogen source

status: Public on Jun 21 2010

city: East Lansing

first: Sean

last: Crosson

state: Michigan

address: 567 Wilson Rd

department: Dept. Microbiology and Molecular Genetics

institute: Michigan State University

summary: This study is measuring the steady-state levels of mRNA in wild-type Caulobacter crescentus grown in M2 defined medium containing either ammonium or nitrate as the sole nitrogen source.

overall_design: Four independent cultures of Caulobacter crecentus were grown in each of two medium conditions: M2(nitrate)glucose and M2(ammonium)glucose.  Cultures in each medium type were grown to OD660=0.3 and RNA was isolated from each.


title: Expression data from cord blood derived CD4+ CD25+ (Treg) and CD4+ CD25- (Thelper) cells, both while resting & after stimulation

city: Adelaide

first: Stephen

middle: Martin

last: Pederson

state: SA

address: North Terrace

department: Biological Sciences

institute: University of Adelaide

summary:
Here we compare the effects of stimulation on cord blood derived CD4+ CD25+ (Treg) and CD4+ CD25- (Thelper) cells, isolated by MACS protocols & expanded in vitro using dynabeads. Expansion was carried out at a ratio of 3 beads/cell in the presence of 1000units/ml of recombinant human IL2 for 8 days, followed by 3 days of culture without beads.
RNA was extracted from resting cells on day 4 after expansion. Stimulated cells were treated with Ionomycin for 2hrs prior to RNA extraction.

overall_design: Four way comparison of resting and stimulated Tregs versus Thelpers. A total of 18 arrays were analysed containing RNA from 5 individual cord blood donors.

first: Tim

last: Sadlon

first: Kaimen

last: Peng

first: M

middle: F

last: Shannon

first: Simon

middle: C

last: Barry


title: Measuring the effects of hypoxia on transcription in Caulobacter crescentus

summary: To test the effects of hypoxia on transcription in Caulobacter crescentus, we cultured cells in a New Brunswick bioreactor under controlled conditions.  Prior to innoculation, the medium was bubbled with laboratory air at maximum flow and stirred at 300 rpm for 2 hours.  After this period, the medium was considered saturated with air and the oxygen probe was set to 100%.  Untreated cultures were grown in air-saturated complex medium at 30 degrees C to OD660=0.5 at pH=7 (continuous air-bubbling; 300 rpm stirring).  At cell harvest in aerated culture, the dissolved oxygen probe remained above 98%.  To subject cells to hypoxia, culture at OD660=0.5, pH=7 was sparged continuously with nitrogen gas; the dissolved oxygen level as measured by the gas probe dropped from 100% to 0% over the course of 5 minutes under this condition.  Hypoxic cultures were continually stirred and bubbled with nitrogen for another 20 minutes after the dissolved gas probe read 0%.  Hypoxic cells were then harvested for RNA isolation.

overall_design: Four independent biological samples are included in this study.  Two batches of cells were subjected to 20 minutes of hypoxia in a bioreactor; two cell batches were highly aerated.


title: RNA expression profiles in CD34+ and in stromal cells of patients with refractory anemia with ringed sideroblasts

city: Sao Paulo

first: Yuri

middle: Barros

last: Moreira

state: SP

address: Av. Prof. Lineu Prestes 748 sala 1200

department: Biochemistry

country: Brazil

institute: Universidade de Sao Paulo

summary: Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Noncoding-RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. In this study, gene expression profiles of CD34+ and stromal cells of MDS patients with refractory anemia with ringed sideroblasts (RARS) subgroup were compared those of healthy individuals, using 44k combined intron-exon oligoarrays, which included probes for protein-coding genes, for sense and antisense strands of totally intronic noncoding (TIN) and for partially intronic noncoding (PIN) RNAs. In CD34+ cells of MDS-RARS patients, 217 genes were significantly differentially expressed (q-value < 0.01) in comparison to healthy individuals, of which 68 (31%) were noncoding transcripts. In stromal cells of MDS-RARS, 13 genes were significantly differentially expressed (q-value < 0.05) in comparison to healthy individuals, of which 4 (30%) were noncoding transcripts. These results demonstrated, for the first time, in CD34+ cells and stromal cells the differential ncRNA expression profile between MDS-RARS and healthy individuals, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

overall_design: Bone marrow (BM) CD34+ cell samples were collected from 4 healthy subjects and from 4 MDS patients. Stromal samples were collected from 4 healthy subjects and 3 MDS patients.  All patients were diagnosed as RARS according to the French-American-British (FAB) classification and did not present chromosomal abnormalities; they received no growth factors or any further MDS treatment.

first: Baratti

middle: O

last: Mariana

first: Moreira

last: Yuri

first: Traina

last: Fabiola

first: Costa

last: Fernando

first: Verjovski-Almeida

last: Sergio

first: Saad

middle: T

last: Sara


title: Aortic arch profiling of Apoe knockout mice

first: Xia

last: Yang

summary: Aortic arch profiling of Apoe-/- mice fed a high fat diet (HFD, Harlan Teklad 88137, 21% fat, 0.15% cholesterol) starting at 8 weeks of age for 8, 16, and 24 weeks.  B6 mice fed HFD for 16 weeks starting at 8 weeks of age were used as controls.

overall_design: Apoe-/- mice were fed a high fat diet (HFD, Harlan Teklad 88137, 21% fat, 0.15% cholesterol) starting at 8 weeks of age for 8, 16, and 24 weeks.  B6 mice fed HFD for 16 weeks starting at 8 weeks of age were used as controls. At each time point, 9 mice were studied. Mice were euthanized at each time point and whole aorta (from aortic root to renal bifurcation) of each mouse was immediately collected and flash-frozen in liquid N2.   Mice were fasted overnight prior to euthanasia.  the aortic arches of three mice in the same study group were pooled, resulting in three pools per study group.


title: Testing the effects of nitric oxide (oxidative) stress on Caulobacter transcription

summary: The goal of this study was to measure the effects of nitric oxide exposure (using DETA NONOate as a nitric oxide donor) on transcription in Caulobacter.

overall_design: Untreated Caulobacter crescentus were grown to a density of 0.3 (at OD660) in PYE medium (pH 7) in rolled culture tubes.  DETA-NONOate treated  Caulobacter crescentus were grown to a density of 0.3 (at OD660), and then treated with 100 mM DETA NONOate for 30 minutes.


title: The dikaryon formation (mating) transcriptome of the basidomycete fungus Coprinopsis cinerea (Coprinus cinereus)

city: Bloomington

first: Claire

last: Burns

state: IN

address: 1001 E 3rd St

institute: Indiana University

summary:
Mating compatibility in C. cinerea is controlled by two loci, A and B.  Following fusion of undifferentiated hyphal cells, a complex program is initiated which results in the maintenance of one nucleus from each parent in every cell (the dikaryon).  We identified downstream targets of the A locus (A on), the B locus (B on), and both loci (Aon Bon) using strains in which the two pathways are activated separately, and a dikaryotic strain.
keywords: Cell type comparison

overall_design: Four biological replicate samples of each experimental strain were taken.  Labelled cDNA was hybridized to arrays in a 2 channel reaction.  The reference sample was an unmated monokaryotic strain.

first: Patricia

last: Pukkila


title: Array CGH analysis of human gastrointestinal stromal tumors

status: Public on Jun 20 2010

city: Sapporo

first: Hiromu

last: Suzuki

state: Hokkaido

address: S1, W17, Chuo-ku

department: Department of Molecular Biology

institute: Sapporo Medical University

summary: Gastrointestinal stromal tumors (GISTs) are the most important mesenchymal tumors of the gastrointestinal tract.  The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown.  In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy. A total of 25 GIST specimens were studied using Human Genome CGH Microarray Kit 105A (G4412A, Agilent).  Levels of LINE-1 methylation were analyzed using bisulfite-pyrosequencing.  LINE-1 hypomethylation was correlated with risk grade, and high-risk GISTs exhibited lower levels of LINE-1 methylation than low- or intermediate-risk GISTs.  Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations.  Our data suggest that LINE-1 hypomethylation correlates with the aggressiveness of GISTs.  Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations.

overall_design: A total of 25 surgically obtained human gastrointestinal stromal tumors (GISTs) was analyzed using Agilent CGH microarray. Copy number aberration was compared with clinicopathological features and DNA methylation status.

first: Minoru

last: Toyota


title: Expression profiling of over-expression of conspecific and heterspecific miR-310 cluster in Drosophila (tiling array)

first: TIAN

last: TANG

summary: The miRNA 310-311-312-313 cluster from D. melanogaster (referred to as Dm310s) and the homolog from D. pseudoobscura (Dp310s) were used to transform D. melanogaster. The over-expression of UAS-miR310s in two Dm310s transgenic lines (M1-7 and M1-3) and Dp310s transgenic line P4-18 was driven by GAL4 line NP5941 with native miR-310 expression pattern. We used Drosophila Tiling 1.0 F arrays to assess the effects of miR310s over-expression on the whole transcriptome.

overall_design:
We compared the transcriptional profiling of Dm310s and Dp310s overexpression in D.melanogaster using double-stranded cDNA followed by bioprime random labeling, and hybridization to Affy Drosophila tiling 1.0 F array. Third-instar progeny larvae from the crosses with maternal NP5941 and paternal UAS-miR-310 cluster transgenic lines were collected and third-instar larvae from the cross with maternal NP5941 and paternal w1118 were used as control. Three biological repeats were used for each stock.
A processed data matrix reporting values (log2 intensity after quantile normalization) for all of the Samples is linked below as a supplementary file.

first: Tian

last: Tang

first: Chung

middle: I

last: Wu


title: Expression profiling of dme-miR-310 cluster (miR-310, miR-311, miR-312, miR-313) knockout

summary: Expression profiling of the dme-miR-310 cluster knockout line in D.melanogaster. The knockout line was generated in an imprecise P-element excision screen using a stock P{EP}2587 containing a P-element insertion directly upstream of the miR-310 cluster. The precise excision line of P{EP}2587 was used as the control.

overall_design:
The expression of miR-310 cluster knockout line (imprecise deletion of P{EP}2587 insertion) were compared with the control (precise deletion of P{EP}2587 insertion) using Drosophila Tiling 1.0 F array. Each stock has three biological repeats.
A processed data matrix reporting values (log2 intensity after quantile normalization) for all of the Samples is linked below as a supplementary file.


title: Microarray analysis of Rgg-dependent transcriptional regulation in Streptococcus suis serotype 2 strain 05ZYH33

city: Nanjing

first: Feng

last: Zheng

address: 293 East Zhongshan Road

institute: Research Institute for Medicine of Nanjing Command

summary: Streptococcus suis 2 Rgg-dependent transcription was analyzed.

overall_design: Microarray analysis was performed using RNA samples isolated from Streptococcus suis 2 wild-type strain 05ZYH33 as well as RNA isolated from 05ZYH33 rgg isogenic mutant strain during postexponential phases of growth.

first: Jiaqi


title: MAQC-II Project: Iconix data set

status: Public on Sep 29 2010

city: Shanghai

first: Leming

last: Shi

address: 2005 Songhu Road

department: School of Life Sciences

institute: Fudan University

type:
Third-party reanalysis
Expression profiling by array

summary:
The Iconix data set (endpoint B) was provided by Iconix Biosciences, Inc. (Mountain View, CA, USA). The study objective was to assess, upon short term exposure, hepatic tumor induction bynon-genotoxic chemicals, since there are currently no accurate and well-validated short-term tests to identify non-genotoxic hepatic tumorigens, thus necessitating an expensive 2-year rodent bioassay before a risk assessment can begin.
The reanalysis has been carried out with log2 transformed gene expression levels of the raw signal values.
The data in this Series were initially submitted in GEO Series GSE8251.

overall_design: The training set consists of hepatic gene expression data from 216 male Sprague-Dawley rats treated for 5 days with one of 76 structurally and mechanistically diverse nongenotoxic hepatocarcinogens and non-hepatocarcinogens. The validation set consists of 201 male Sprague-Dawley rats treated for 5 days with one of 68 structurally and mechanistically diverse non-genotoxic hepatocarcinogens and non- hepatocarcinogens. Gene expression data were generated using the Amersham Codelink Uniset Rat 1 Bioarray (GE HealthCare, Piscataway, NJ). The separation of the training set and validation set was based on the time when the microarray data were collected; i.e., microarrays processed earlier in the study were used as training and those processed later were used as validation.


title: RamA as a global regulator for gene expression in Salmonella Typhimurium

city: college park

first: jie

last: zheng

state: MD

address: 5100 paint branch parkway

department: division of microbiology

institute: center for food safety and applied nutrition, FDA

summary: RamA, a 113-amino-acid regulatory protein, belongs to the AraC-XylS transcriptional activator family. Here, the network of genes regulated by RamA was investigated by determining changes in transcript profiles when ramA gene was knocked out from wild type LTL strain, which expresses ramA constitutively.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: Analysis used aerobic log-phase state RNA from S. Typhimurium strain LTL (ciprofloxacin MIC: 4 µg/ml) derived from S. Typhimurium LT2 by in vitro-induction using ciprofloxacin as control samples for comparison to the experimental samples taken from its mutant strain, LTLvramA with ramA-deletion. Six biological replicates were used for the experiment.Two replicates for each sample. One replicate per array.

first: Jie

first: Fei

last: Tian

first: Shenghui

last: Cui

first: Jiuzhou

last: Song

first: Eric

middle: W

last: Brown

first: Jianghong

last: Meng


title: WSC1extMID2cyt strain 2 hour exposure to caspofungin

status: Public on Jun 19 2010

city: Madrid

first: Javier

last: Arroyo

state: Madrid

address: Plaza Ramon y Cajal S/N

department: Microbiologia II

country: Spain

institute: Facultad de Farmacia (UCM)

summary:
We did transcription profiling on the effect of wsc1 deletion, gene involved in cell wall stress response
Keywords: cell wall stress response

overall_design: S. cerevisiae (WSC1extMID2cyt strain BY4741) was grown on YEPD. For caspofungin experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 15ng/ml of caspofungin. Cells were collected at 2 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.

first: Clara

last: Bermejo

first: Raul

last: Garcia

first: Andrea

last: Straede

first: Jose

middle: M

last: Rodriguez-Peña

first: Cesar

last: Nombela

first: Jürgen

last: Heinisch


title: MID2extWSC1cyt strain 2 hour exposure to caspofungin

overall_design: S. cerevisiae (MID2extWSC1cyt strain BY4741) was grown on YEPD. For caspofungin experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 15ng/ml of caspofungin. Cells were collected at 2 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.


title: mid2 mutant 2 hour exposure to caspofungin

overall_design: S. cerevisiae (mid2 mutant strain BY4741) was grown on YEPD. For caspofungin experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 15ng/ml of caspofungin. Cells were collected at 2 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.


title: sho1 mutant 3 hour exposure to zymolyase

summary:
We did transcription profiling on the effect of sho1 deletion, gene involved in cell osmotic stress and cell wall stress response
Keywords: cell wall stress response

overall_design: S. cerevisiae (sho1 mutant) was grown on YEPD. For Zymolyase experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 0.8 units/ml Zymolyase 100T. Cells were collected at 3 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.


title: Wsc1 mutant 4 hour exposure to Congo Red

overall_design: S. cerevisiae (wsc1 mutant strain BY4741) was grown on YEPD. For Congo Red experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 30 μg/ml of Congo Red. Cells were collected at 4 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.


title: wsc1 mutant 3 hour exposure to zymolyase

summary:
We did transcription profiling on the effect of wsc1 deletion, gene involved in cell osmotic stress and cell wall stress response
Keywords: cell wall stress response

overall_design: S. cerevisiae (wsc1 mutant) was grown on YEPD. For Zymolyase experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 0.8 units/ml Zymolyase 100T. Cells were collected at 3 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.


title: mid2 mutant 4 hour exposure to Congo Red

summary:
We did transcription profiling on the effect of mid2 deletion, gene involved in cell wall stress response
Keywords: cell wall stress response

overall_design: S. cerevisiae (mid2 mutant strain BY4741) was grown on YEPD. For Congo Red experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 30 μg/ml of Congo Red. Cells were collected at 4 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.


title: Wsc1 mutant 2 hour exposure to caspofungin

overall_design: S. cerevisiae (wsc1 mutant strain BY4741) was grown on YEPD. For caspofungin experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 15ng/ml of caspofungin. Cells were collected at 2 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.


title: Insulin-like Signaling Determines Survival During Stress via Post Transcriptional Mechanisms in C. elegans.

status: Public on Jun 17 2010

city: Novato

first: Simon

last: Melov

address: 8001 Redwood Blvd

institute: Buck Institute for Research on Aging

summary: The insulin-like signaling (ILS) pathway regulates metabolism and is known to modulate adult lifespan in C. elegans. Altered stress responses and resistance to a wide range of stressors are also associated with changes in ILS and contribute to enhanced longevity. The transcription factors DAF-16 and HSF-1 are key effectors of the longevity phenotype. We demonstrate that increased intrinsic thermotolerance, due to lower ILS, is not dependent on stress induced HSF-1 transcriptional responses but instead requires active protein translation. Translation profiling experiments reveal genes that are post-transcriptionally regulated in response to altered ILS during heat shock in a DAF-16-dependent manner. Furthermore, several novel proteins are specifically required for ILS effects on thermotolerance. We propose that lowered-ILS results in DAF-16-induced metabolic and physiological changes that precondition a translational response to modulated survival under acute stress.

overall_design: 72 experimental samples were analyzed using custom oligo microarrays. A wild type sample pool was used as the Cy3 reference/control for all experimetal samples. All extracted RNA prior to array analysis was fractioned (via a sucrose gradient) based on ribosomal loading and pooled into ribosomal and free RNA (F1), light polysomes (F2) and heavy polysomes (F3) as described in the experimental procedures. The control RNAi is ‘empty’ vector L4440 RNAi feeding vector plasmid (1999 Firelab vector kit) transformed HT115(DE3), which was obtained from the Caenorhabditis Genetics Center (University of Wisconsin).

first: Gawain

last: McColl

first: Aric

middle: N

last: Rogers

first: Silvestre

last: Alavez

first: Alan

last: Hubbard

first: Christopher

middle: D

last: Link

first: Ashley

last: Bush

first: Pankaj

last: Kapahi

first: Gordon

last: Lithgow


title: Rainbow trout erythrocytes stimulated with LPS and Poly(I:C)

city: Cerdanyola del Vallès

first: Davínia

middle: Morera

last: Capdevila

address: Edifici Biociències, ala sud

department: Biologia cel.lular, Immunologia i Fisiologia

institute: Universitat Autònoma de Barcelona

summary: The focus of this study was to investigate the response of cultured rainbow trout erythrocytes to lipopolysaccharide (LPS) and to the analog viral dsRNA Poly(I:C) using a salmonid-specific microarray platform enriched with immune-related genes. Although the transcriptomic response measured as the total number of differentially expressed genes was higher in LPS, the intensity response, in terms of genes with a FC>2, was greater in Poly(I:C) treated cells. These two treatments shared the regulation of some genes, but not alls followed the same pattern. Over representation of Gene Ontology functional categories showed us that Poly(I:C) treatment produced a immune response whereas LPS stimulation affect biological processes specially.

overall_design: Trout erythrocytes were isolated from blood by twice Ficoll 1.007 density gradient centrifugations. For primary cell culture, erythrocytes were resuspended in DMEM (PAA Laboratories) supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories) and the antibiotic Primocin (100 μg/ml, Invivogen) at a density of 7.5x106 cells/ml in six well cell culture plates (NUNC) and cultured at 18 ºC and 5% CO2. Erythrocytes were stimulated with LPS from E. coli (serotype 026:B6, Sigma) and Poly(I:C) (Invivogen) at 50 μg/ml, each treatment had their own control plate. All culture plates were incubated for 24h. Total RNA was extracted from the cultures using 1 ml of Tri Reagent (Sigma) following the manufacturer’s instructions with minor modifications. Quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes.

last: Morera

last: Mackenzie


title: mid2 mutant 3 hour exposure to zymolyase

first: Javier

last: Arroyo

summary:
We did transcription profiling on the effect of mid2 deletion, gene involved in cell osmotic stress and cell wall stress response
Keywords: cell wall stress response

overall_design: S. cerevisiae (mid2 mutant) was grown on YEPD. For Zymolyase experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 0.8 units/ml Zymolyase 100T. Cells were collected at 3 hours of growth, frozen at -80 °C and processed for RNA extraction. The total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.

last: Bermejo

last: Garcia

middle: J


title: Molecular signature of mineralocorticoid signalling in cardiomyocytes

status: Public on Jun 18 2010

city: Nantes

first: Marja

last: Steenman

address: 8 quai Moncousu

institute: L'institut du thorax

summary: Gene expression analysis of hearts from double transgenic mice with conditional, cardiomyocyte-specific, overexpression of the mineralocorticoid (MR) or of the glucocorticoid receptor (GR).

platform_organism:
Homo sapiens
Mus musculus

overall_design: GR-mice vs controls, MR-mice vs controls. GR: 3 sample pools hybridized to 2 microarrays each. MR: 1 sample pool hybridized to 3 microarrays. Microarray contains triplicate spots.


title: T7-SAGE transcriptome of cleft versus end bud epithelial cells of embryonic mouse salivary glands

city: Suita

first: Takayoshi

last: Sakai

state: Osaka

address: 1-8 Yamadaoka

department: Oral-Facial Disorders

institute: Osaka Univ Grad Sch Dent

summary: Mus musculus embryonic submandibular salivary glands from embryonic day 12.5 embryos were laser microdissected to isolate epithelial cells from regions adjacent to forming clefts or from the peripheral end bud region. The isolated region-specific samples were then subjected to T7-SAGE transcriptome analysis.

overall_design: Toluidine blue-stained cryostat sections of embryonic day 12.5 submandibular salivary glands were used for laser microdissection using an LMD system (Leica) with a pulsed UV laser on an upright automated microscope. A total of approximately 500 cells from a region immediately adjacent to a forming cleft or from a peripheral end bud region were excised by the laser and transferred to caps of 0.5 ml PCR tubes.  SAGE libraries were constructed from the microdissected cleft and bud epithelia cells.  SAGE tags were identified using the National Center for Biotechnology Information (NCBI) tag map set for UniGene.


title: Expression analysis of human adipose tissue stem cells (ADSCs) transduced with homeobox C8 (Hoxc8) or control vector, followed by treatment with adipogenic-induction media for 14 days

city: SUita

first: Masaki

last: Mori

address: 2-2 Yamada-oka

department: Graduate School of Medicine

institute: Osaka University

summary: Adipogenesis occurs through a specific gene program in undifferentiated fat progenitors. We hypothesized that the properties of the fat progenitors are regulated by hox genes, the developmental genes essential in different tissue stem cells. Their biased expression in white and brown fat implies roles in distinguishing the two fat types. Among 39 Hox genes, Hoxc8 is highly enriched in undifferentiated adipose tissue stem cells (ADSCs) and down-regulated in differentiated adipocytes. Forced expression of Hoxc8 suppressed adipocyte differentiation of ADSCs. Using microarrays, we investigated the effect of Hoxc8 overexpression on global transcripts in ADSCs. We compared among four groups: untreated ADSCs, adipogenic induction media (MDI)-treated ADSCs, MDI-treated ADSC-vector and MDI-treated ADSC-Hoxc8. A number of, but not all, adipogenesis-related genes are suppressed by Hoxc8. This dataset illustrates the global effect of Hoxc8, a developmental transcription factor, on the expression of adipogenesis-related genes.

overall_design:
Gene expression was compared among untreated ADSCs (control), adipogenic induction media-treated ADSCs, adipogenic induction media-treated ADSC-vector (ADSCs transduced with control vector), and adipogenic induction media-treated ADSC-Hoxc8 (ADSCs transduced with human Hoxc8).

Total RNA was isolated from ADSCs using the Qiagen RNeasy kit (Qiagen). At NimbleGen, quality and yield were verified before cDNA synthesis and Cy3-end labeling. The labeled cDNA samples were hybridized to Homo sapiens 4-Plex arrays (Roche NimbleGen, A4487001-00-01) that represent 24,000 human genes. Raw data files for each sample were normalized and background-corrected using a Robust Multi-Array Analysis as implemented by NimbleScan software. Students’ two-tail t-tests were conducted among the samples for each transcript and fold-change was determined. Transcripts whose abundance was significantly altered (P < 0.05) and an absolute fold change greater than 2 were defined as differentially regulated.

first: Hironori

last: Nakagami

first: Yasufumi

last: Kaneda