first: Andre

last: Gerber

summary:
To identify mRNAs associated with human PUM1 protein, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip) approach on HeLa S3 cancer cells that express PUM1. PUM ribonucleoprotein (RNP) complexes were captured from cell-free extracts with anti PUM1 specific antibody Bethyl Laboratories, #300-201A coupled to protein G (PUM1) sepharose beads, and then eluted with SDS-EDTA. To control for non-specifically enriched RNAs, the same procedure was performed with beads that were not coupled with immunoprecipitating antibody (mock samples). Total RNA was isolated from cell extracts and immunopurified samples with the mirVanaTM PARISTM kit (Ambion). RNA was quantified with a NanoDrop device (Witeg AG). Poly-adenylated RNAs were amplified in the presence of aminoallyl-UTP with Amino Allyl MessageAmp II aRNA kit (Ambion). For this purpose, 500 ng total RNA from extracts and half (50-100 ng) of the immunopurified RNAs were used for amplification. 8 ug of the amplified RNAs (aaRNA) were fluorescently labeled with NHS-monoester Cy3 and Cy5 dyes (GE HealthSciences), except for mock RNA samples, where an aaRNA amount proportional to the yield obtained from corresponding PUM affinity isolates was used. For PUM1 RIPs, we performed three biological replicates with technical (dye swap) replicates (total six arrays).(~40 ug aaRNA from PUM1 RIPs, ~9 ug aaRNA from mock RIPs). The Cy3- and Cy5-labeled aaRNA samples were mixed and hybridized to human cDNA microarrays.Detailed methods for microarray experiments are available at http://cmgm.stanford.edu/pbrown/protocols/index.html. cDNA microarrays were produced by the Stanford Functional Genomic Facility and contained 43,197 human probes representing 26,524 Unigene cluster IDs (12,466 ENSEMBL annotated genes) spotted on Corning Ultra GAPS slides. Spotted cDNAs were cross-linked with 65 mJ of UV irradiation on slides, which were then post-processed for 1 hour at 42°C in pre-hybridization solution (5x SSC, 0.1% SDS, 0.1 mg/ml BSA), washed twice in 400 ml of 0.1x SSC for 5 min, dunked in 400 ml ultrapure water for 30 sec, and dried by centrifugation at 550 rpm for 5 min. Slides were used the same day. Cy3- and Cy5-labeled aaRNA probes were mixed and applied to arrays in hybridization solution (3x SSC, 20 ug poly(A) RNA [Invitrogen], 20 ug yeast tRNA [Invitrogen], 20 ug Human Cot-1 DNA [Invitrogen], 20 mM HEPES [pH 7.0] and 0.3% SDS) for 18 h at 65°C. The arrays were then washed sequentially in 400 ml of 2x SSC with 0.1% SDS, 1x SSC, and 0.2x SSC. The first wash was performed for 5 min at 65°C, the subsequent washes were performed for 5 min at RT. The arrays were dried by centrifugation and immediately scanned with an AxonScanner 4200A (Molecular Devices). Data were collected using GENEPIX 5.1 (Molecular Devices). Arrays were normalized computationally by the Stanford Microarray Database (SMD). The data were filtered for signal over background of greater than 1.5 in the channel measuring aaRNA from extract, and only features that met these criteria in > 50% of the arrays were included for further analysis. Log2 median ratios were retrieved and exported into Microsoft Excel.To identify transcripts that were specifically enriched by association with PUM1, we performed two class Significance Analysis of Microarrays (SAM) on median centered arrays.
A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments.
Antibody used in IP: 20 ug goat anti-PUMILIO 1 Bethyl Laboratories, #300-201A

Keywords: replicate_design

first: Alessia

last: Galgano


status: Public on Sep 17 2008

city: Heidelberg

first: Alberto

last: Calabro

address: INF 580

country: Germany

institute: DKFZ

summary:
Comparison between Estrogen receptor positive and Estrogen receptor negative breast cancer samples
Keywords: breast cancer type comparison

overall_design: 152 unique breast cancer tissue sample are included in the analysis. The total mRNA has been labeled with Cy5 and then hybridized on a two color arrays against the stratagen Human common reference that was previously labelled with Cy3.

first: Michael

last: Stojanov

first: Holger

last: Sueltmann

first: Annemarie

last: Poustka


title: Microarray-Bioinformatics analysis of altered genomic expression profiles in the human fetal and infant myocardium

status: Public on Sep 01 2008

city: Beijing

first: bo

last: kong

address: 167 Beilishi Road

country: China

institute: Cardiovascular Institute and Fuwai Hospital,CAMS&PUMC

summary:
Develop of the heart during perinatal period must involve the differentially expression of a myriad of genes. We can think about the developmental events of heart formation in the context of genetic networks. However the knowledge of the genetic basis in governing and regulating of this development is still partial and scattered; an overall elucidation of the differential expressed genes blueprint before and after birth has never been proposed. The global recognition of difference in molecular level between the fetus and infants is necessary for it can provide not only further revealing the mechanism of heart development, but also set up a better molecular foundation of myocardial protection in fetal cardiac surgery for pediatric cardiac surgeons. In this study we outline a genetic blueprint for the development between fetal and postnatal heart using DNA microarray, and we also validate the microarray result by RT-PCR. Comparing with the microarray results from fetal and natal heart, The differentially expression gene was analyzed by gene ontology (GO), Entra Gene and can be partitioned to 15 categories. The meanings of some categories had been discussed. The differentially genes include the groups for metabolism, hyperplasia, development, extracellular matrix components, transcription regulation, signal transduction et al relative. It is remarkable that we also found some differentially expression genes never reported before, that will provide some clue for future heart development research.
Keywords: fetal,infant,myocardium,Bioinformatics,differentially expressed gene profile

overall_design: We analyzed differentially expressed gene profiles of 5 pairs of of human fetal and infant myocardium samples by the DNA microarrays provide by CapitalBio corporation, validate the microarray result by RT-PCR , classified altered expressed genes in all the 5 groups and elucidated the biological process of these genes in more details with the aid of bioinformatics

first: Bo

last: Kong

first: Yinglong

last: Liu

first: Xiaodong

last: Lv


title: Maintenance of undifferentiated mouse ES cells in suspension by the serum and feeder-free defined culture condition

status: Public on Sep 02 2008

city: Baltimore

first: Minoru

last: Ko

state: MD

address: 251 Bayview Blvd, Suite 100, 10C

department: National Institute on Aging

country: USA

institute: NIH

summary:
Mouse embryonic stem (ES) cells are indispensable for gene targeting
approaches to study gene functions and regulations, the production of animal models for human diseases, and in vitro cell differentiation studies. The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium is free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified.  DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions and activation of the WNT signaling pathway. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products.
Keywords: reference design,replicate design

overall_design: RNA was extracted by using Isogen (Wako) according to the manufacturer's instructions.  First-strand cDNA was prepared by using Super Script reverse transcriptase (Invitrogen) primed with random hexamer in a 20ml reaction mixture containing 5 mg of total RNA.  A total of 0.5 ml of the first-strand cDNA mixture was used for PCR with Taq polymerase (TAKARA) performed in a 50-ml volume.  The following conditions were used: 10 min at 95C followed by 40 cycles of 15 seconds at 95C and 1 min at 60C (for Oct4, Nanog, ERas, Rex-1, Snail1, Gata4, Slug, and Gapdh) or 2 min at 94C followed by 25 cycles of 30 seconds at 94C, 30 seconds at 56C, and 1 min at 72C (for Itgb8, Cdh11, and Col3a1).  The PCR reaction products were separated by gel electrophoresis, and the DNA bands were visualized under ultraviolet light for photography.

first: Yukiiko

last: Tsuji

first: Naoko

last: Yoshimura

first: Hitomi

last: Aoki

first: Alexei

middle: A

last: Sharov

middle: S

first: Tsutomu

last: Motohashi

first: Takahiro

last: Kunisada


title: E2F in vivo binding specificity: comparison of consensus vs. non-consensus binding sites

status: Public on Oct 01 2008

city: Davis

first: Alina

last: Rabinovich

state: CA

address: 451 Health Science Dr.

department: Genome Center

institute: UC Davis

type: Genome binding/occupancy profiling by genome tiling array

summary:
	Recent genome-scale ChIP-chip studies of transcription factors have shown that a low percentage of experimentally determined binding sites contain the consensus motif for the immunoprecipitated factor.  In most cases, differences between in vivo target sites that contain or lack a consensus motif have not been explored.  We have previously shown that most sites to which E2F family members are bound in vivo do not contain E2F consensus motifs. The main purpose of this study was to develop an understanding of how E2F binding specificity is achieved in vivo. In particular, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif.  Using promoter and ENCODE arrays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are a) the site must be in a core promoter and b) the promoter region must be utilized as a promoter by the transcriptional machinery in that particular cell type.  We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including a) indirect recruitment, b) looping to the core promoter mediated by an E2F bound to a distal consensus motif, and c) assisted binding of E2F to a site that weakly resembles an E2F consensus motif within the core promoter.  To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated promoter fragments.  Our findings suggest that in vivo a) the presence of a consensus motif is not sufficient to recruit E2Fs, b) E2Fs can bind to isolated regions that lack a consensus motif, and c) binding can require regions other than the best match to the E2F PWM in the core promoter.
Keywords: E2F, ChIP-chip, transcription factor binding, consensus motifs

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
37 ChIP-chip arrays (of these, 14 array sets are biological duplicates).
22 samples are included in this series, the rest can be found in supplementary info to the following papers: Xu 2007, Jin 2006, Komashko 2008

first: Victor

middle: X

last: Jin

first: Roman

first: Xiaoqin

last: Xu

first: Peggy

middle: J

last: Farnham


title: Gene expression profiling in human fibroblast after low-LET irradiation

city: Montreal

first: Olivier

middle: Charles

last: Maes

state: Quebec

address: 3999 Cote Ste Catherine

department: Bloomfield Centre for Research in Aging

country: Canada

institute: Lady Davis Institute for Medical Research

summary: Exposure to radiation provokes cellular responses controlled in part by gene expression networks. MicroRNAs (miRNAs) are small non-coding RNAs which mostly regulate gene expression by degrading the messages or inhibiting translation. Here, we investigated changes in miRNA expression patterns after low (0.1 Gy) and high (2.0 Gy) doses of X-ray in human fibroblasts. At early (0.5 h) and late (6 and 24 h) time points, irradiation caused qualitative and quantitative differences in the down-regulation of miRNA levels, including miR-92b, 137, 660, and 656. A transient up-regulation of miRNAs was observed after 2 h post-irradiation following high doses of radiation, including miR-558 and 662. MicroRNA levels were inversely correlated with targets from mRNA and proteomic profiling after 2.0 Gy of radiation. MicroRNAs miR-579, 608, 548-3p, and 585 are noted for targeting genes involved in radioresponsive mechanisms, such as cell cycle checkpoint and apoptosis. We suggest here a model in which miRNAs may act as "hub" regulators of specific cellular responses, immediately down-regulated so as to stimulate DNA repair mechanisms, followed by up-regulation involved in suppressing apoptosis for cell survival. Taken together, miRNAs may mediate signaling pathways in sequential fashion in response to radiation, and may serve as biodosimetric markers of radiation exposure.

overall_design: The gene expression patterns in human fibroblasts after 2.0 Gy of low-LET radiation was determined at  2 and 24 hrs post-irradiation time in technical triplicates. Control non-irradiated samples were also prepared in triplicates.

middle: C

first: Eugenia

last: Wang


title: Karyotype analysis of S. cerevisiae chromosome replacement lines

status: Public on Sep 16 2008

city: Taipei

first: Jun-Yi

last: Leu

address: 128 Academia Road, Section 2, Nankang

department: Insititute of Molecular Biolody

country: Taiwan

institute: Academia Sinica

type: Genome variation profiling by genome tiling array

summary: This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Saccharomyces bayanus
Saccharomyces cerevisiae

overall_design: Refer to individual Series


title: Analysis of genes associated with MYC in maintaining tumorigenesis

status: Public on Aug 28 2008

city: Stanford

last: Stanford Microarray Database

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

summary:
The MYC oncogene has been implicated in the regulation of up to thousands of genes involved in many cellular programs including proliferation, growth, differentiation, self-renewal, and apoptosis. MYC is thought to induce cancer through an exaggerated effect on these physiologic programs. Which of these genes are responsible for the ability of MYC to initiate and/or maintain tumorigenesis is not clear. Previously, we have shown that upon brief MYC inactivation, some tumors undergo sustained regression. Here we demonstrate that upon MYC inactivation there are global permanent changes in gene expression detected by microarray analysis. By applying StepMiner analysis, we identified genes whose expression most strongly correlated with the ability of MYC to induce a neoplastic state. Notably, genes were identified that exhibited permanent changes in mRNA expression upon MYC inactivation. Importantly, permanent changes in gene expression could be shown by chromatin immunoprecipitation (ChIP) to be associated with permanent changes in the ability of MYC to bind to the promoter regions. Our list of candidate genes associated with tumor maintenance was further refined by comparing our analysis with other published results to generate a gene signature associated with MYC-induced tumorigenesis in mice. To validate the role of gene signatures associated with MYC in human tumorigenesis, we examined the expression of human homologs in 273 published human lymphoma microarray datasets in Affymetrix U133A format. One large functional group of these genes included the ribosomal structural proteins. In addition, we identified a group of genes involved in a diverse array of cellular functions including: BZW2, H2AFY, SFRS3, NAP1L1, NOLA2, UBE2D2, CCNG1, LIFR, FABP3, and EDG1. Hence, through our analysis of gene expression in murine tumor models and human lymphomas, we have identified a novel gene signature correlated with the ability of MYC to maintain tumorigenesis.
Time: time of doxycycline treatment or time after removal

Keywords: time_series_design

overall_design:
Groups of assays that are related as part of a time series.
Compound Based Treatment: Bone tumor cells 1325 were treated/not treated with 20ng/ml of doxycycline
Computed

first: Natalie

last: Wu


title: Cocaine Regulates MEF2 to Control Synaptic and Behavioral Plasticity

status: Public on Aug 24 2008

city: Boston

first: Will

last: Renthal

state: MA

address: 60 Fenwood Rd.

institute: Brigham and Women's Hospital

summary: Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a novel mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine, and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine.

overall_design: Mice were treated for 7 days with daily injections of cocaine (20 mg/kg) and sacrificed 24 hrs later. Chromatin from bilateral punches of NAc was immunoprecipitated with an antibody against MEF2A as described previously with minor modifications (Renthal et al., 2007). Chromatin was sonicated to an average of ~500 bp and immunoprepitated with antibody against MEF2A (Santa Cruz, sc-313) or an IgG control (Upstate/Millipore).  Antibody-bound chromatin was precipitated using Protein A beads from Upstate (06-157), which were washed with low salt, high salt, and LiCl buffers to remove non-specific DNA binding. Eluted chromatin was reverse-crosslinked at 65oC in the presence of proteinase K and EDTA. DNA was purified by chloroform extraction/ethanol precipitation and the enrichment of specific promoters was amplified by ligation-mediated PCR for genome-wide analysis (Sikder et al., 2006).  Amplified DNA was then labeled with Cy3 (input-enriched) or Cy5 (MEF2-enriched) and hybridized to Nimblegen (Madison, WI) MM8 mouse promoter arrays. Bilateral nucleus accumbens from eight mice were pooled for microarray analysis.


title: Sall4 regulates distinct transcription circuitries in different blastocyst-derived stem cell lineages

status: Public on Aug 19 2008

city: Singapore

first: Bing

last: Lim

state: Singapore

address: 60 Biopolis Street

department: Stem Cell and Developmental Biology

country: Singapore

institute: GIS

summary:
Stem cells self-renew or differentiate under the governance of a stem cell-specific transcriptional program with each transcription factor orchestrating the activities of a particular set of genes. Here we demonstrate that a single transcription factor is able to regulate distinct core circuitries in two different blastocyst-derived stem cell lines, embryonic stem (ES) and extra-embryonic endoderm (XEN) cells. The transcription factor, Sall4, is required for early embryonic development and for ES cell pluripotency. Sall4 is also expressed in XEN cells and depletion of Sall4 disrupts self-renewal and induces differentiation. Genome-wide analysis reveals Sall4 is regulating different gene sets in ES and XEN cells, and depletion of Sall4 targets in the respective cell types induces differentiation. With Oct4, Sox2 and Nanog, Sall4 forms a crucial interconnected auto-regulatory network in ES cells. In XEN cells, Sall4 regulates key XEN lineageassociated genes, Gata4, Gata6, Sox7 and Sox17. Our findings demonstrate how Sall4 functions as an essential stemness factor for two different stem cell lines.

Keywords: ES/XEN comparison, ES Sall4 KD/control KD comparison, and XEN Sall4 KD/control KD comparison

overall_design:
In this study, we delineated the core circuitries that are regulated by Sall4 in governing the distinctive blastocyst-derived stem cell lines. We dissected crucial components of the Sall4-driven circuitries through loss-of-function analyses, and showed that these are required for maintaining the stem cell states.

Six gene expression experiments have been performed: ES wildtype, ES Sall4 KD, ES control KD, XEN wildtype, XEN Sall4 KD and XEN Sall4 control KD. Each experiment has 3 replicates (see Samples GSM313602..GSM313619). Data were normalized with cross-correlation method; differential expression was based on the mean of the log2 fold changes (see supplementary files below).

ChIP-chip experiments have also been performed (see Samples GSM319610..GSM320264).

first: Chin

middle: Y

first: Wai-Leong

last: Tam

first: Jinqiu

last: Zhang

first: Haw

last: Ang

first: Hui

last: Jia

first: Leonard

last: Lipovich

first: Chia-Lin

last: Wei

first: Wing

middle: K

last: Sung

first: Paul

last: Robson

first: Henry

last: Yang


title: Wild-type versus HBx-transfected mive

status: Public on Sep 11 2008

city: Seoul

first: Seong

last: Je kyung

country: South Korea

institute: Seoul National University

summary:
Hepatitis B virus (HBV) has been clearly recognized as an etiological factor for hepatocellular carcinoma (HCC). HBV encodes the potentially oncogenic HBx protein. We aimed to elucidate the molecular mechanism of HCC caused by HBx and to discover the biomarker related to HCC by HBx. Three experimental groups, 3, 9 and 13 month aged HBx Tg mice and age matched normal wild type B6 mouse which have same background of HBx Tg mice were used to find differentially expressed genes during HCC.

Keywords: Genetic modification

overall_design: 3-month-old, 9-month-old, 13-month-old wild type B6 mice vs 3-month-old, 9-month-old, 13-month-old HBx transfected mice; Biological replicates at each timepoint; 9 controls vs 9 HBx-mice

first: Je-kyung

last: Seong


title: Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells

status: Public on Sep 13 2008

first: Minoru

last: Ko

summary:
We showed the function of Rex1 in mouse ES cells as well as in embryos using the conventional gene targeting strategy. Our results clearly indicated that Rex1 function is dispensable for both the maintenance of pluripotency in ES cells and the development of embryos. However, Rex1-/- ES cells showed the defect to induce a subset of the marker genes of visceral endoderm, when differentiated as embryoid body, as found in EC cells. Conclusion: Rex1 should be regarded just as a marker of pluripotency without functional significance like the activity of alkaline phosphatase.

Keywords: genetic modification design,reference design,replicate design

overall_design:
We established ES cell lines with four different genotypes for Rex1; ES cells carrying the wild-type Rex1 alleles and the empty CAG-IZ vector (wt), the wild-type Rex1 alleles and the Rex1 transgene (wt-Tg), the Rex1-/- alleles and the empty vector(KO), and the Rex1-/- alleles and the Rex1 transgene (KO-Tg). The genetically-engineered ES cell lines were generated to analyze the function of Rex1 in the maintenance of pluripotency and to analyze its gain- and loss-of function. For loss-of-function analysis, we disrupted the endogenous Rex1 allele by conventional gene targeting via homologous recombination in ES cells. The knock-out (KO) allele should be a functionally null allele because the first 100 bp of the open reading frame in the exon 4 including the start codon was replaced by the pacEGFP chimeric gene cassette containing the puromycin-resistant gene (pac) and the green fluorescent protein (Egfp) cDNA. Interestingly, all of the puromycin resistant clones obtained by transfection of this KO vector carried the correctly targeted alleles. One of the Rex1+/- ES cell line (RKPG9) was cultured with high-dose puromycin to obtain the Rex1-/- ES cell lines generated via spontaneous gene conversion. Multiple Rex1-/- ES cell lines were established with extremely high efficiency (4 of 4 clones obtained after the selection were homozygous for Rex1 KO allele). Correct targeting events were confirmed by the loss of the polymorphic signature of the wild-type allele on the southern blot analysis of the genomic DNA, in which the 5.6 kb fragment corresponds to the Rex1 pseudogene on chromosome 15 reported previously as well as found in the mouse genome data. Northern blot revealed the loss of the transcript derived from the wild-type allele in Rex1-/- ES cells, which express the large transcripts composed by the truncated Rex1 and pacEGFP. Rex1-/- ES cells were also established by introduction of the second knockout vector carrying the hygromycin-resistant gene as a selection marker.

Rex1+/- ES cell line (RKPG9), Rex1-/- ES cells (HP3 and HP4), and one wild-type ES cells (EB5)

first: Shinji

last: Masui

first: Satoshi

last: Ohtsuka

first: Rika

last: Yagi

first: Kadue

last: Takahashi

first: Hotoshi

last: Niwa


title: mRNAs associated with human PUM1 and PUM2 protein

status: Public on Sep 08 2008

city: ETH Hoenggerberg, Zuerich

first: Andre

last: Gerber

address: Wolfgang Pauli Str. 10, HCI H396

department: Institute of Pharmaceutical Science

country: Switzerland

institute: Swiss Federal Institute of Technology


title: Effect of microgravity on brain gene expression in mice

status: Public on Aug 08 2008

city: Prairie View

first: Dumitru

middle: Andrei

last: Iacobas

state: TX

address: Ann Preston St

department: Undergraduate Medical Academy

institute: Prairie View A&M University

summary: Changes in gravitational force such as that experienced by astronauts during space flight induce a redistribution of fluids from the caudad to the cephalad portion of the body together with an elimination of normal head-to-foot hydrostatic pressure gradients. To assess brain gene profile changes associated with microgravity and fluid shift, a large-scale analysis of mRNA expression levels was performed in the brains of 2 weeks control and hindlimb-unloaded (HU) mice using cDNA microarrays. Although to different extent, all functional categories displayed significantly regulated genes indicating that considerable transcriptomic alterations are induced by HU. Interestingly, the TIC class (transport of small molecules and ions into the cells) had the highest percentage of up-regulated genes, while the most down-regulated genes were those of the JAE class (Cell junction, Adhesion, Extracellular Matrix). TIC genes comprised 16% of those whose expression was altered, including sodium channel, nonvoltage-gated 1 beta (Scnn1b), glutamate receptor (Grin1), voltage-dependent anion channel 1 (Vdac1), calcium channel beta 3 subunit (Cacnb3) and others. The analysis performed by GeneMAPP revealed several altered protein classes and functional pathways such as blood coagulation and immune response, learning and memory, ion channels and cell junction. In particular, data indicate that HU causes an alteration in hemostasis which resolves in a shift toward a more hyper-coagulative state with an increased risk of venous thrombosis. Furthermore, HU treatment seems to impact on key steps of synaptic plasticity and learning processes.

overall_design: We used brains of four control (C1, C2, C3, C4) and four tail-suspended hindlimb-unloaded (E1, E2, E3, E4) mice to ensure statistical relevance of the study. 60 ug of total RNA extracted in TRIzol from each brain was reversed transcribed into labeled cDNAs using fluorescent Cy5 or Cy3-dUTP (Amersham Biosciences, NJ). Differently labeled cDNAs obtained from a pair of biological replicas were co-hybridized overnight at 50C with a microscope slide spotted with ~ 27,000 mouse cDNA sequences produced by the Microarray Core Facility of the Albert Einstein College of Medicine in the “multiple yellow” design (i.e.: C1C2, C3C4, E1E2, E3E4), described in Iacobas DA, Fan C, Iacobas S et al., Transcriptomic changes in developing kidney exposed to chronic hypoxia. Biochem Biophys Res Commun. 2006 349(1):329-38).

first: Antonio

last: Frigeri

first: Sanda

first: Grazia

middle: P

last: Nicchia

first: Jean

middle: F

last: Desaphy

first: Diana

last: Camerino

first: Maria

last: Svelto

first: David

last: Spray


title: MF1 outbred stock liver gene expression data

status: Public on Aug 22 2008

city: los angeles

first: anatole

last: ghazalpour

address: 675 charles young dr south

institute: UCLA

summary:
Linkage analysis of complex traits in mice is a powerful tool to find loci affecting the phenotype but it has a poor resolution making it difficult to identify the underlying genes. We show here, using whole genome association analysis of gene expression traits in an outbred mouse population, the MF1 stock, that mapping resolution is greatly increased as compared to linkage. The fact that eQTLs discovered in other crosses were replicated and successfully mapped with high resolution in this population provides a strong proof of concept. In addition, we show that this population is a useful resource to resolve the eQTL hotspots detected in other studies. Finally, we highlight the importance of correcting for population structure in whole genome association studies in the outbred stock.
Keywords: genetic association study in outbred mice

overall_design: a total of 110 MF1 mice were used for this study. The gene expression data is from the liver of 8 month old mice. These mice were fed a chow diet till 4.5 months of age and subsequently were fed high fat diet for 14 weeks.

first: Anatole

last: Ghazalpour

first: Sudheer

last: Doss

first: Alec

last: Brozell

first: Hyun

last: Kang

first: Ruth

last: Castellanos

first: Eleazar

last: Eskin

first: Desmond

last: Smith

first: Thomas

last: Drake

first: Aldons

last: Lusis


title: Molecular prognosis in multiple myeloma

status: Public on Aug 05 2008

city: Saint Herblain

first: Wilfried

last: Gouraud

address: bd Jacques Monod

department: Integrated Center of Oncology René Gauducheau

country: France

institute: ICO (Integrated Center of Oncology Nantes)

summary:
Purpose
Survival of patients with multiple myeloma is highly heterogeneous from periods of few weeks to more than ten years. We used gene-expression profiles of myeloma cells obtained at diagnosis to identify broadly applicable prognostic markers.
Methods
In a training set of 182 patients, we used supervised methods to identify individual genes associated with length of survival. A survival model was built from these genes. The validity of our model was assessed in our test set of 68 patients and in three independent cohorts comprising 853 multiple myeloma patients.
Results
The 15 strongest genes associated with the length of survival were used to calculate a risk score and to stratify patients in low-risk and high-risk. The survival-predictor score was significantly associated with survival in both the training and test sets and in the external validation cohorts. The Kaplan Meier estimates of rates of survival at 3 years were 90.5 percent (95 percent CI, 85.6 – 95.3), and 47.4 (95 percent CI, 33.5 - 60.1) respectively in our patients having a low-risk or high-risk, independently of traditional prognostic factors.
High-risk patients constituted a homogeneous biological entity characterized by the overexpression of genes involved in cell cycle progression and its surveillance, while low-risk patients were heterogeneous and displayed hyperdiploid signatures.
Conclusion
Gene expression-based survival prediction and molecular features associated with high-risk patients may be useful for developing prognostic markers and may provide basis to treat these patients with new targeted anti-mitotics.
Keywords: Gene-expression profiling

overall_design: 250 myeloma patients at diagnosis examined with spotted cDNA nylon membrane

first: Olivier

last: Decaux

first: Laurence

last: Lodé

first: Florence

last: Magrangeas

first: Catherine

last: Charbonnel

first: Pascal

last: Jézéquel

first: Philippe

last: Moreau

first: Jean-Luc

last: Harousseau

first: Michel

last: Attal

first: Régis

last: Bataille

first: Loïc

last: Campion

first: Hervé

last: Avet-Loiseau

first: Stéphane

last: Minvielle


title: Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size

status: Public on Jul 23 2008

city: Little Rock

first: Radhakrishnan

last: Nagarajan

state: AR

address: 4301 W Markham, Slot 781, Room: 3234

department: Department of Biostatistics

institute: University of Arkansas for Medical Sciences

summary: The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-month) and old (32-month) male Brown Norway/F344 rats by two weeks of hind limb suspension (HS) and soleus muscles were analyzed by cDNA microarrays. We conclude that a cold shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss.

overall_design: 6 (C3, Cy5) arrays along with their dye-swap were generated for the aged rats; 3 (C3, Cy5) arrays along with their dye-swap were generated for the adult rats. Each array had two replicates top and bottom consisting of the same 9984 genes each.

first: Esther

middle: E

last: Dupont-Versteegden


title: Gene expression profiles of human promyelocytic leukemia cell lines exposed to six volatile organic compounds

first: Sailendra

middle: Nath

last: Sarma

address: P.O. Box 131, Cheongryang

institute: Korea Institute of Science and Technology

summary: The gene expression profiles of HL-60 cells were determined for two doses (IC20 and IC50) of three chemicals (ethylbenzene, tricholoretylene and dichloromethane) and for the IC50 doses of 3 other chemicals (benzene, toluene and o-xylene) at a 3 h time point. Assays were performed in triplicate for each chemical/dose, and individual gene-expression profiles were determined for each chemical at the tested concentration doses.

overall_design: Gene expression analysis was conducted on the RNA samples using 35-K whole human genome microarray (Operon Biotechnologies, Inc. Germany). Three independent experiments were performed for each chemical (N = 6), simultaneously. Labeling and hybridization were performed using the instructions of the Platinum Biochip Reagent Kit (GenoCheck Co. Ltd., Korea). This was followed by the coupling of the Cy3 dye for the controls (DMSO) and Cy5 dye for the treated samples. Hybridization was performed in a hybridization oven at 62 oC for 12 h. After washing (2× SSC/0.1% SDS for 2 min at 58 oC, 1× SSC for 2 min at RT and 0.2× SSC for 3 min at RT), the slide was dried by centrifugation at 800 rpm for 3 min at RT. Hybridization images on the slides were scanned by ScanArray Lite (PerkinElmer Life Sciences, USA). Scanned images were analyzed with GenePix 3.0 software (Axon Instruments, USA) to obtain gene expression ratios.

middle: N

first: Youn-Jung

last: Kim

first: Jae-Chun

last: Ryu


title: Comparison of WT Vs NaS1-/- mouse ileum

city: St Lucia

first: Sean

middle: M

last: Grimmond

state: QLD

address: Research Rd

department: Genomics and Bioinformatics

country: Australia

institute: Institute of Molecular Bioscience, Uni of QLD

summary:
Pairwise comparison of ileum gene expression in pools (n=14) of wildtype 4 week old male Vs NaS1-/- knockout mice.
Keywords: Genetic modification

overall_design: pairwise comparison of pooled WT Vs pooled NaS1-/- ileum tissues. Samples were hybridized in quadruplicate and 2 sets of samples were dye swapped.

first: Paul

last: Dawson

first: Brooke

last: Gardiner

first: Michael

last: Mcguckin

first: Daniel

last: Markovich


title: Comparison of endocrine enriched genes in islet beta cells versus induced beta cells

status: Public on Jul 08 2008

city: Cambridge

first: qiao

last: zhou

address: 7 Divinity Ave

department: Stem cell and regenerative biology

institute: Harvard University

summary: Endocrine enriched genes in adult islet beta cells were identified and compared with that of induced beta cells (with M3 transcription factors) in adult.  The control sample is non-beta pancreatic cells.

overall_design: Gene expression profile comparison of 3 samples, 3 independent repeats for each sample indicate a high degree of similarity between endogenous and induced beta cells in adult mouse.

first: Qiao

last: Zhou


title: Global gene expression profile of small intestine from wild-type and CFTR-knockout mice.

city: Cincinnati

first: Gary

last: Shull

state: OH

address: 231 Albert Sabin Way ML0524

institute: University of Cincinnati

summary:
The objective of this study was to identify changes in gene expression levels between wild-type and CFTR-knockout small intestine.  CFTR-knockout mice (provided by Dr. Lane Clarke of the University of Missouri) were maintained on colyte.
Keywords: gene expression comparison

overall_design: Four wild-type and four CFTR-knockout small intestinal RNA samples were compared.  To facilitate statistical analysis and reduce affects of Cy3 and Cy5 labeling, comparison of two WT and two KO were repeated with a dye flip.

first: Emily

last: Bradford


title: Gene expression profiling in rat renal allografts after prolonged cold ischemia

status: Public on Jul 10 2008

city: Cologne

first: Bernhard

last: Gerstmayer

address: Stockheimer Weg 1

institute: Miltenyi Biotec GmbH

summary:
Ischemia/reperfusion injury (IRI) has a major impact on the long-term outcome of renal allografts. However, the mechanisms of IRI related to chronic allograft nephropathy (CAN) are still poorly understood. To address this issue, in a F344 to Lewis transplantation model of the rat, kidneys were either subjected to 20 min. or 24 hours of cold ischemia. A customized cDNA microarray representing 737 immune related genes was used for gene expression analysis after 12 hours and 6 months of engraftment. Thereby showing the 6-month graft function and histology were only moderately deteriorated following short cold ischemic time (20 min.), while prolonged ischemia (24 hrs) accelerated CAN development. Following prolonged cold ischemia already 12 hrs after engraftment about 30 genes were differentially expressed up to >20-fold. This included adhesion molecules, heat shock proteins, transcription factors and chemokines (CXCL10) associated with a strong CD68+ macrophage infiltration. Furthermore we observed a remarkable up-regulation of immunoproteasomes implying a re-organisation of the proteasome complex. This data might explain the higher incidence of alloreactivity following enhanced IRI. After 6 months, the prolonged cold ischemia of 24 hours revealed the significant induction of 4 genes: clusterin, C4, and the CCR7 ligands CCL19/21. This supported the observation of enhanced vasculopathy, humoral alloreactivity and increased tracking of MHC-II+ antigen presenting cells during CAN suggesting them as suitable novel targets for combating IRI.
Keywords: transplantation, cold ischemia, inflammation, gene expression profiling

overall_design: Renal allografts from F344 donors were transplanted into unmodified LEW recipients. Before harvesting, grafts were perfused with University of Wisconsin solution at 4°C undergoing 20 min. (group A) or 24 hrs (group B) cold ischemia. Engrafted organs were either removed after 12 hrs (group I-A (n=3) and I-B (n=2)) or after 6 months (group II-A (n=3) and II-B (n=2)). Recipient animals of the group II received CyA treatment for 10 days at a dosage of 1.5 mg/kg. Untreated normal kidneys from F344 rats served as control. RNA from transplanted and untreated (control) rat kidneys were labeled by reverse transcription with Cy5 and Cy3 fluorescence, respectively.

first: K

last: Kotsch

first: P

last: Martins

first: R

last: Klemz

first: U

last: Janssen

first: B

first: A

last: Dernier

last: Reutzel-Selke

last: Kuckelkorn

first: S

middle: G

last: Tullius

first: H-

middle: D

last: Volk


title: Effect of neurotrophin p75 receptor (p75NTR) on angiogenesis

status: Public on Jul 17 2008

city: Amsterdam

first: Anton

last: Horrevoets

address: van der Boechorststraat 7

department: Molecular Cell Biology

country: Netherlands

institute: VU University Medical Center

summary:
Neurotrophins (NTs) promotes angiogenesis and EC survival, via tropomyosin kinase trkA and trkB receptors. A different p75NTR receptor of NTs, which belongs to the TNF-alfa receptor superfamily, is not or scarcely expressed by endothelial cells (EC) and endothelial progenitor cells (EPC) under basal conditions. Both diabetes and muscular ischemia induce p75NTR in capillary EC. In this study, by gene transfer, we forced the expression of p75NTR in EC and EPC to study the effect on cell survival, proliferation, adhesion, migration, and capillary-like tubes formation on matrigel, which all resulted impaired by p75NTR. We identified that p75NTR inhibits the VEGF-A/Akt/eNOS/NO pro-angiogenesis/pro-EC survival pathway and reduces the mRNA contents of survivin and securin in EC. By Illumina technology and real-time PCR, we found that p75-NTR alters the expression of VEGF-A and beta-1 integrin, which are implicated in angiogenesis and cell survival. p75NTR transfer to ischemic murine limb muscles impaired neoangiogenesis and blood flow recovery and induced apoptosis of bone marrow Sca-1+/Lin- progenitor cells. Diabetes induced p75NTR in bone marrow Sca-1+/Lin- cells and this correlated with apoptosis. Finally, inhibition of p75NTR signaling in diabetic ischemic limb muscles restored proper muscular neovascularization and blood flow recovery.
Keywords: Response to ectopic receptor expression on angiogenesis

overall_design: Two series of 4 mice each were treated with either control adenovirus (AdNull) or adenovirus expressing neurotrophin p75 receptor (AdP75). Anaesthetized mice received 3 adenovirus injections (for a total of 109 p.f.u. virus in 20 micro L) into 3 equidistant sites of the normoperfused or ischemic left adductor muscles, as described (2.	Emanueli C, Graiani G, Salis MB, Gadau S, Desortes E, Madeddu P. Prophylactic gene therapy with human tissue kallikrein ameliorates limb ischemia recovery in type 1 diabetic mice. Diabetes. 2004 Apr;53(4):1096-103. )

first: Costanza

last: Emanueli


title: Global gene expression profile of small intestine from wild-type and NHE3-knockout mice.

summary:
The objective of this study was to identify changes in gene expression levels between wild-type and NHE3-knockout small intestine.
Keywords: gene expression comparison

overall_design: Six wild-type and six NHE3-knockout small intestinal RNA samples were compared.  To facilitate statistical analysis and reduce affects of Cy3 and Cy5 labeling, three sets were done with reciprocal dye flips.


title: RAW264.7 cells were treated with KDO, IFN beta and 8Br

status: Public on Jun 28 2008

city: Pasadena

first: Sangdun

last: Choi

address: 1200 E. California Blvd

department: Biology

institute: California Institute of Technology

summary:
RAW 264.7 cells were stimulated with KDO (100 ng/ml), IFN-β (300 pM) and/or 8-Br 100 μM. These ligands were applied individually or in combination with KDO for 60 min and 120 min. Total RNA was extracted using TriPure (Roche) following its protocol.
Keywords: designed

overall_design:
RAW 264.7 cells were stimulated with KDO (100 ng/ml), IFN-β (300 pM) and/or 8-Br 100 μM. These ligands were applied individually or in combination with KDO for 60 min and 120 min. Total RNA was extracted using TriPure (Roche) following its protocol. The detailed procedure is available at http://www.signaling-gateway.org/data/ProtocolLinks.html (AfCS Procedure Protocol PP00000009). Duplicate experiments were done for each treatment.

Oligonucleotide array fabrication and annotation
16 K mouse oligonucleotide arrays were fabricated with 15,631 oligomers of 65-bp or 70-bp long. The oligomers were purchased from Operon and Sigma-Genosys and were inkjet-printed onto glass slides by Agilent Technologies. The list of genes is available through the GEO (Gene Expression Omnibus - http://www.ncbi.nlm.nih.gov/geo) as an accession number GPL254.

Gene expression analysis
 The hybridization experiment and analysis were performed as previously described (Park et al., 2004; Zhu et al., 2006; Zhu et al., 2004). Cy5-labeled cRNA (RNA of ligand treated cells) and Cy3-labeled cRNA (RNA of time matched controls) were hybridized in the array. Dye-swap labeling was performed for each pair of samples. The arrays were scanned with Agilent Scanner G2505A (Agilent Technologies) and image files were extracted with background subtraction and dye-normalization using Agilent G2566AA Extraction Software version A.6.1.1 (Agilent Technologies).

first: Rebecca

last: Hart

first: MiSook

last: Chang

first: Robert

last: Hsueh

first: Jong-Woo

last: Kim

first: YunAnna

last: Cao

first: SunYoung

last: Lee

first: Angela

last: Alexander

first: Joella

last: Grossoehme

first: Zhen

last: Yan


title: Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors

status: Public on Jun 30 2008

city: Paris

first: Stéphane

last: LE CROM

address: 46 rue d'Ulm

department: Biology Institute - IBENS

institute: École normale supérieure

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Melan-a melanocytes and B16 melanoma cells

status: Public on Jul 31 2008

city: Huntington

first: James

last: Denvir

state: WV

address: One John Marshall Drive

department: Biochemistry and Microbiology

institute: Marshall University School of Medicine


title: Expression and genomic changes after exposing drug-selected mutants to short term CQ treatment in Plasmodium falciparum.

status: Public on Jun 26 2008

city: Rockville

first: Hongying

last: Jiang

address: 12735 Twinbrook Parkway

institute: NIAID/NIH

type:
Expression profiling by array
Genome variation profiling by array

summary:
Mutations in PfCRT confer chloroquine (CQ) resistance in P. falciparum. Point mutations in the homolog of the mammalian multidrug resistance gene (pfmdr1) can also modulate the levels of CQ response. However, parasites with the same pfcrt and pfmdr1 alleles exhibit a wide range of drug sensitivity, suggesting that additional genes contribute to levels of CQ resistance (CQR).
We used 3 isogenic lines which have different drug resistance profiles corresponding to unique mutations in the pfcrt gene (106/1K76, 106/176I, and 106/76I-352K) to study changes in gene expression with and without CQ and genomic variations, i.e. copy number (CN) changes.
RNA transcription levels from 45 genes were significantly altered in one or both mutants relative to the parent line. Of particular interest are genes encoding proteins involved in transport and/or regulation of cytoplasmic or compartmental pH, e.g. the V-type H+ pumping pyrophosphatase 2, Ca2+/H+ antiporter VCX1 and copy number changes in pfmdr1. A series of deletion (including 15 genes) also occurred at the beginning of chromosome 10.
Keywords: low-dose 3h Chloroquine response, copy number variation

platform_organism:
Plasmodium falciparum
Anopheles gambiae

overall_design: Three isogenic parasite lines were treated with and without 3h CQ and synchronized for total RNA extraction and hybridization to Affymetrix GeneChips® to study gene expression profiles. Genomic DNA from mixed culture was also extracted and hybridized to the same Affymetrix GeneChips. Total 18 total RNA samples (3 biological replicates per condition) and 6 genomic DNA samples (2 biological replicates per condition).

first: Jigar

last: Patrel

first: Ming

last: Yi

first: Jianbing

last: Mu

first: Jinhui

last: Ding

last: Stephens

first: Roland

middle: A

last: Cooper

first: Michael

middle: T

last: Ferdig

first: Xinzhuan

last: Su


title: Time course RA-treatment of B16 mouse melanoma cells

summary:
Time course experiment for treatment of B16 mouse melanoma cells with all-trans retinoic acid

Keywords: Time couse

overall_design: Time-course experiment with treatment at 4hr, 10hr, 24hr, and 48hr. Six biological replicates per time point; one biological replicate per array. Dye swap.

first: Mary

last: Estler

first: Goran

last: Boskovic

first: Sarah

last: Miles

first: Don

last: Primerano

first: Richard

last: Niles


title: Genomic expression profiling of TNFalpha treated BDC2.5 diabetogenic CD4+ T Cells

status: Public on Jul 01 2008

city: Stanford

first: Li-Fen

last: Lee

address: 299, Campus Dr.

department: Microbiology and Immunology

institute: Stanford University

summary:
Tumor necrosis factor alpha (TNFalpha) plays an important role in immune regulation, inflammation, and autoimmunity.  Chronic TNFalpha exposure has been shown to down-modulate T cell responses.  In a mouse T cell hybridoma model, TNFalpha attenuated T cell receptor (TCR) signaling.  We have confirmed that chronic TNFalpha and anti-TNFalpha exposure suppressed and increased T cell responses in BDC2.5 CD4+ T cells, respectively.  The goal of this study is to analyze global transcriptional alterations resulting from TNFalpha treatment on TCR signaling pathways using cDNA microarrays.  We found that genes involved in functional categories including T cell signaling, cell cycle, proliferation, ubiquitination, cytokine synthesis, calcium signaling, and apoptosis were modulated.  Genes such as ubiquitin family genes, cytokine inducible SH2-containing genes, cyclin-dependent kinase inhibitors p21, p57, calmodulin family genes (calmodulin -V1, -V2, and ?V3) and calcium channel voltage- dependent, N type alpha1B subunit (CaV2.2) were induced by TNFalpha, while Vav2, Rho GTPase activating protein, calcium channel voltage dependent, L type alpha 1C subunit (CaV1.2), interleukin-1 (IL) receptor-associated kinase 1, and -V2 (IRAK-1and -2) and IL enhancer binding factor 3 were reduced by TNFalpha.  Genes such as CaV1.2 and proliferating cell nuclear antigen, repressed by TNFalpha, were induced by anti-TNF treatment.  Further, we showed that chronic TNFalpha exposure impaired NF-kappaB and AP-1 transactivation activity, leading to T cell unresponsiveness.  Thus, our results present a detailed picture of transcriptional programs affected by chronic TNFalpha exposure, and provide candidate target genes, which may function to mediate TNFalpha induced T cell unresponsiveness.
Set of arrays that are part of repeated experiments
Keywords: Biological Replicate

overall_design: The experiments addressed the effect of TNF exposure.  8-12-week-old NOD.BDC2.5 transgenic mice were injected i.p. with PBS or 3 ug murine TNFalpha; on alternate days for three weeks.  At day 24 of treatment, suspensions of LNs and/or splenocytes were stimulated with the indicated concentration of the mimotope (1047-7 peptide (YVAPVWVRME)) at a density of 7-8 x 106 cells/ml, in a final volume of 1 ml in 24-well plates.  After prolonged in vitro culture, cells were harvested 12 hrs post transplantation.  CD4+ T cells were purified by labeling of monoclonal Abs of surface markers, Vbeta4-FITC and CD4-PE and enriched by Magnetic beads selection followed by FACS sorting.  The purity of CD4+ T cells ranged from 95% to 99% depending on individual CD4 T cells preparation.  The data in column represents six independent repeated experiments with half of the slides being reverse replicates.  We compared RNA from PBE treated group with TNF treated group without including reference RNA.

first: Jason

last: Lih


title: Gene expression and lung tumors: screening for markers that discriminate tumor histology and genetic background.

status: Public on Jul 22 2008

city: Madrid

first: Barbara

last: Angulo

address: Melchor Fernandez Almagro, 3

department: Molecular Pathology Program

country: Spain

institute: Spanish National Cancer Centre

summary:
The success of targeted therapies creates a need to discriminate tumors accurately by their histological and genetic characteristics. Here, we used cDNA microarray analysis to identify gene expression profiles and single markers that recapitulate the pathological and genetic background (i.e., BRAF, EGFR, KRAS, LKB1, PIK3CA, and TP53 gene alterations) of non-small cell lung cancer (NSCLC).
Gene expression profiles were determined for 6 normal lung tissues and 69 lung tumors from patients diagnosed with NSCLC. We performed an unsupervised hierarchical clustering with the most variably expressed transcript to investigate whether there was evidence for natural grouping samples based on similarity in gene expression profiles. We examined the relationship between the clusters of tumors and patient and tumor characteristics such as gender, smoking status, histological type, degree of differentiation, tumor size, lymph node involvement and genetic background. We used a supervised analysis to identify the genes that are important for distinguishing subgroups of tumors defined by histological type. Moreover, we used this supervised approach to search for markers that characterize those tumors carrying EGFR, KRAS, PIK3CA and TP53 alterations.
We identify several genes with characteristic patterns of expression in each tumor histological type (adenocarcinoma and squamous cell carcinoma) and we found that the presence of EGFR mutations result in a particular expression profile.
Keywords: Transciption profiling of tumors with different histological and genetic background.

overall_design: We analyzed 69 NSCLC tumors. All the samples were characterized by histological type and by the presence of alterations at genes that are known to be involved in lung carcinogenesis. Mutational analysis of BRAF, EGFR, KRAS, and LKB1 is provided only for adenocarcinomas and analysis of alterations at PIK3CA gene (mutational analysis or gene amplification by FISH analysis) is provided only for 47 of the tumors. We analyzed gene expression profiles to identify those genes that are differentially expressed between two subgroups by t-test: 1) adenocarcinomas vs. squamous cell carcinomas; 2) tumors with mutation at EGFR gene vs. wild type tumors; 3) tumors with mutation at KRAS gene vs. wild type tumors; 4) tumors with mutation at TP53 gene vs. wild type tumors; 5) tumors with alterations (mutation or gene amplification) at PIK3CA gene vs. wild type tumors.

first: Ana

last: Suarez-Gauthier

first: Montserrat

last: Sanchez-Cespedes


title: Transcriptome Study of CD34+ hematopoietic progenitor from patients reached of Primary Myelofibrosis

status: Public on Jul 29 2008

city: VILLEJUIF

first: christophe

last: desterke

address: 14-16 avenue Paul Vaillant Couturier

department: UFR medecine institute Andre Lwoff

institute: university Paris Sud11

summary: An increased number of circulating CD34+ hematopoietic progenitors (HP) and a prominent amplification of  dystrophic  megakaryocytes (MK) are observed in PMF patients. As transcriptome data from CD34+ hematopoietic progenitors showed modulations of FLT3 and MAP kinase expression independently of the JAK2V617F mutation status

overall_design:
Transcriptome analysis was performed on circulating CD34+ cells from PMF patients using Agilent 22K microarray and compared to CD34+ cells from blood and bone marrow from un-mobilized healthy donors.
Indirect map: each tested sample was hybridzed with reference probe

first: Christophe

last: Desterke

first: Vladimir

last: Lazar

first: Bernadette

last: Guerton

first: Olivier

last: Pierre-Louis

first: Denis

last: Clay

first: Stephane

last: Giraudier

first: Chrystèle

last: Bilhou-Nabéra

first: Marie-Caroline

last: Le Bousse-Kerdilès


title: Effects of Aging and Calorie Restriction on the Global Gene Expression Profiles of Mouse Testis and Ovary

status: Public on Jun 09 2008

city: Baltimore

first: Minoru

last: Ko

address: 251 Bayview Blvd, Suite 100, 10C

department: National Institute on Aging

institute: NIH

summary:
AGEMAP is an initiative of the National Institute on Aging (NIA) designed to study the aging process on mice with Caloric Restriction (CR) versus Ad Libitum (AL) diet. The aim of the AGEMAP project is to provide a valuable source of information about genetic influences involved in aging of the major organ systems. A total of 67 mice (C57BL/6) were sacrificed. Both male and female mice were divided in 7 categories based on the diet and the age. Four categories in ad libitum (1 month, 6 month,16 month and 24 month) and three categories in caloric restriction (6 month, 16 month and 24 month). In the current study we focused on gonads, using microarray hybridization (Agilent platform 44K array) to identify gene expression changes age-associated CR-associated, between young versus old mice at different time points.
Keywords: development or differentiation design,replicate design,stimulus or stress design,time series design

overall_design:
The experiment was done as time-course type. Four time points for mice on ad libitum diet (1 month, 6 month, 16 month and 24 month), and three time points on caloric restriction diet. For each time point we used two biological replicates.
We analyzed gene expression patterns in ovaries and testes sampled from 1-, 6-, 16-, and 24-month-old C57BL/6 mice fed on AL and CR diets (Figure 1A and D). Mice were kept on an AL diet until 14 weeks of age and then split into AL and CR gonads . Two individual animals for each condition were used for the microarray studies. We examined a total of 28 samples (two AL ovaries at each of 1, 6, 16, and 24 months; two CR ovaries at 6, 16, and 24 months; two AL testes at 1, 6, 16, and 24 months; and two CR testes at 6, 16, and 24 months). These 28 samples were hybridized on whole-genome oligonucleotide microarrays bearing approximately 44,000 probes, representing 25,585 nonredundant genes with gene symbols  (Additional file 1). The same RNA samples were used for quantitative reverse-transcription polymerase chain reaction (RT-PCR)

first: Alexei

last: Sharov

first: Geppino

last: Falco

first: Yulan

last: Piao

first: Suresh

last: Poosala

first: Kelvin

last: Becker

first: Alan

middle: B

last: Zonderman

first: Dan

middle: L

last: Longo

first: David

last: Schlessinger

middle: S


title: BAF250B-associated SWI/SNF chromatin-remodeling complex is required to maintain undifferentiated mouse ES cells

summary:
We used BAF250b-/- ES cells, two independently derived clones, at 18 and 72 hr in culture and compared them with parental cell line (wild-type) at the same time in culture (two replications each). Total RNAs were extracted using Trizol (Invitrogen) according to manufacturer protocol. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference RNA (Stratagene). Labeled targets were purified using an RNeasy Mini Kit (Qiagen) according to the Agilent's protocol, quantitated by a NanoDrop scanning spectrophotometer (NanoDrop Technologies), and hybridized to the NIA Mouse 44K Microarray v2.2 (whole genome 60-mer oligo; manufactured by Agilent Technologies, #014117) (Carter et al. 2005) according to the Agilent protocol (G4140-90030; Agilent 60-mer oligo microarray processing protocol - SSC Wash, v1.0). All hybridizations were carried out in the two color protocol by combining one Cy3-CTP-labeled experimental target and Cy5-CTP-labeled reference target. Microarrays were scanned on an Agilent DNA Microarray Scanner, using standard settings, including automatic PMT adjustment.
Keywords: genetic modification design,time series design

overall_design: Biological replications were performed on BAF250b-/- ES cells with their parental line (wild-type) at 18 and 72 hrs after passaging, as well as with 12 other pluripotent cells (ES and EG cells from strains C57BL/6 and 129 data from Sharova et al., 2007) using whole-genome mouse microarrays (NIA 44k  V2.2 with 60-mer synthesized oligos).

first: Zhijiang

last: Yan

first: Zhong

last: Wang

first: Lioudmila

last: Sharova

first: Chen

last: Ling

first: Kazuhiro

last: Aiba

first: Ryo

last: Matoba

first: Weidong


title: Gene expression analysis upon exposure of hESCs to novel set of self-renewal and differentiation compounds

status: Public on Jun 05 2008

city: New York

first: Agnes

last: Viale

state: NY

address: 1275 York Avenue

department: Molecular Biology

institute: Memorial Sloan Kettering Cancer Center

summary:
Here we present a strategy to adapt hESCs to high-throughput screening (HTS) conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several currently marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice. Global gene expression analysis upon drug treatment reveals overlapping and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the available repertoire of chemical compounds for manipulating hESC fate.
Keywords: Global gene expression analysis, drug response, human ES cells, pluripotency, differentiation, self-renewal

overall_design: Undifferentiated hESCs were exposed to control and drug treatments for 24 hours followed by mRNA expression analysis

first: Sabrina

middle: C

last: Desbordes

first: Lorenz

last: Studer


title: DNA methylation, H3K4me2, H3K27me3, Pol2 promoter profiling during neuronal differentiation

status: Public on Jun 04 2008

city: Basel

first: Fabio

last: Mohn

address: Maulbeerstrasse 66

institute: Friedrich Miescher Institute for Biomedical Research

type: Methylation profiling by genome tiling array

summary:
Cellular differentiation entails loss of pluripotency and parallel gain of lineage-specific and ultimately cell-type specific characteristics. Using a murine system that progresses from stem cells to lineage-committed progenitors and further to terminally differentiated neurons we analyzed two repressive epigenetic pathways: DNA methylation and Polycomb-mediated methylation of histone H3 (H3K27me3). We show that several hundred promoters become DNA methylated in lineage-committed progenitor cells. Targets are selected for pluripotency and germline-specific genes, suggesting a role for DNA methylation in stabilizing loss of pluripotency already at the progenitor state. Conversely, we detect loss and acquisition of H3K27me3 at novel targets at both progenitor and terminal state. Surprisingly, many neuron-specific genes that are poised to be activated upon terminal differentiation become Polycomb targets only in progenitor cells. Moreover, promoters marked by H3K27me3 in stem cells frequently become DNA methylated during differentiation, suggesting context-dependent crosstalk between Polycomb and DNA methylation. This data suggest a new model how de novo DNA methylation and dynamic switches in Polycomb targets restrict pluripotency and define the developmental potential of progenitor cells.

Keywords: MeDIP-chip, ChIP-chip, neuronal differentiation time-course

overall_design: MeDIP-chip and ChIP-chip experiments were performed with at least two independnet biological replicates. For each condition hybridizations include a dye-swap experiment.


title: Gene expression changes in the intestinal crypts of mice with loss of imprinting

status: Public on Jul 16 2008

first: Andrew

last: Feinberg

address: 720 Rutland Ave., 1064 Ross

department: Medicine

institute: Johns Hopkins University

summary:
To identify genes differentially expressed in the intestinal crypts between LOI(+) and LOI(-) mice, we performed  microarray experiments using RNA extracted from laser capture microdissection, by microdissecting an average of 8,000 crypts from each of 3 LOI(+) and 3 LOI(-) mice.
Keywords: genetic modification design

overall_design: H19 mutant mice with C57BL/6J background carrying a deletion in the H19 gene (3 kb) and 10 kb of the upstream region including differentially methylated region (DMR) were maintained without LOI phenotype by breeding female wild-type C57BL/6J and male H19+/-. Experimental crosses were performed between female H19+/- and male wild-type C57BL/6J to obtain LOI(+) mice and LOI(-) mice. Igf2 on the maternal allele is silenced in LOI(-), i.e. wild type, mice. But when H19 DMR deletion is inherited from mother, the normally silenced Igf2 on maternal allele is activated and LOI (loss of imprinting) of Igf2 occurs. LOI(+) and LOI(-) mice were sacrificed at 120 days, and the tissue pieces of the small intestine were collected from the middle (the third fifth) part and kept frozen at -80C. To detect gene expression change in intestinal progenitor cells clearly, Laser Capture Microdissection (LCM) was performed to collect intestinal crypt cells. The 10 um thick sections were prepared from the frozen small intestine pieces, and 8,000 intestinal crypts at average were dissected by LCM for each of three LOI(+) and LOI(-) mice, and 2 ug of total RNA at least were collected using RNeasy Kit (GIAGEN). 1.7 ug of total RNA for each sample were used for labeling to compare gene expressions in intestinal crypt between LOI(+) and LOI(-) mice.

first: Atsushi

last: Kaneda

first: Chiaochun

middle: J

first: Raymond

last: Cheong

first: Patrick

last: Onyango

first: Bo

last: Wen

first: Christine

last: Iacobuzio-Donahue

first: Rolf

last: Ohlsson

first: Rita

last: Andraos

first: Mark

last: Pearson

first: Alexei

last: Sharov

first: Minoru

last: Ko

first: Andre

last: Levchenko


title: E-cadherin and EMT regulation by bHLH factors: a new role for the class I gene E2-2 (TCF4) and its comparison with E47.

status: Public on Dec 21 2008

city: Bron

first: Hector

last: Hernandez-Vargas

state: Rhone

address: 36 bis Rue de la Batterie

institute: Genomics Consulting

summary: Loss of expression of the cell-cell adhesion molecule E-cadherin is an essential event for epithelial-mesenchymal transition (EMT), a process that allows cell migration in the developing embryo and during tumour invasion. Transcriptional repression has emerged as the main mechanism responsible for E-cadherin downregulation in most carcinomas. Recently, we have identified the class I HLH transcriptional regulator E2-2 (ITF2), in a yeast one hybrid screen designed to identify transcriptional repressors interacting with the E-pal element of the murine E-cadherin promoter. The E2-2 gene codifies two isoforms that differ in their N-terminal regions but their specific functions remain unknown. In the present work we show that both E2-2A and E2-2B induce a complete EMT, in MDCK cells, with loss of E-cadherin expression, gain of mesenchymal markers and acquisition of motile and invasive properties. Although both isoforms repress E-cadherin promoter in MDCK cells, only the E2-2B isoform does it in other epithelial cell lines. Notably, we found that E2-2B is upregulated at the mRNA level in MDCK cells after treatment with TGF-beta1, a key regulator of EMT. Upregulation of E2-2A/B factors was confirmed in MDCK cells overexpressing other EMT inducers, Snai1, Snai2 or E47. Interestingly, gene profiling studies indicate that bHLH E2-2 factors induce similar, yet distinct, genetic programs from those induced by bHLH E47 in MDCK cells. These results, together with the expression pattern observed in early mouse embryos, support a new role for E2-2A/B in E-cadherin regulation and EMT, and suggest an interesting interplay between bHLH factors and other E-cadherin repressors.

overall_design: The Oncochip microarray platform v2.0 contains 13,824 clones printed by duplicate  corresponding to 9,300 different genes. Each of the MDCK transfectants (E22A, E22B) were labeled with dUTP-Cy5 and hybridized against the dUTP-Cy3-labeled MDCK-CMV controls. One additional hybridizations using reciprocal fluorochrome labeling were performed (dye-swap) in each clone. Thus, a total of two hybridizations were performed for each condition.

first: Verónica

middle: R

last: Sobrado

first: Liam

last: Holt

first: Gema

last: Moreno-Bueno

first: Angela

last: Nieto

first: Francisco

last: Portillo

first: Amparo

last: Cano


title: Analysis of gene expression in different T cell clones

status: Public on May 30 2008

city: Napoli

first: Raffaele

last: De Palma

address: via Pansini 5

department: Clinical & Experimental Medicine

country: Italy

institute: Second University of Naples

summary:
The aim was to analyze the expression of genes in T cell clones having different functional profiles (Th1, Th0, Th17, Th2) obtained from the same individual.

Keywords: Gene expression, T cell clones, Th17 characterisation

overall_design: The T cell clones were derived according to well known protocol, using different cytokines, and classified on the basis of their ability to produce cytokines. Once characterized for their production of IL-4, IL-12, gamma-IFN, IL-13, IL-1, they were used to prepare microarray experiments.

first: Gabriella

last: Rodolico

first: Lorenzo

last: Cosmi

first: Francesco

last: Annunziato


title: Transcriptome-wide noise controls differentiation potential of mammalian progenitor cells

status: Public on May 22 2008

city: Boston

first: Hannah

middle: H.

last: Chang

address: 1 Blackfan Circle

department: Vascular Biology

institute: Childrens Hospital Boston, Harvard Medical School

summary:
Phenotypic cell-to-cell variability within clonal populations may be amanifestation of "gene expression noise", or it may reflect stablephenotypic variants.  Such "non-genetic cell individuality" can arisefrom the slow fluctuations of protein levels in mammalian cells. Thesefluctuations produce persistent cell individuality, thereby rendering aclonal population heterogeneous. However, it remains unknown whetherthis heterogeneity may account for the stochasticity of cell fatedecisions in stem cells which depends on the kinetics that underliesheterogeneity.  Here we show that in clonal populations of hematopoieticprogenitor cells, spontaneous "outlier" cells with extremely high or lowexpression levels of the stem cell marker Sca-1 reconstitute theparental distribution of Sca-1 but do so only after more than one week.This slow relaxation is described by a Gaussian-Mixture Model (GMM) thatincorporates noise-driven transitions between discrete subpopulations,suggesting hidden multi-stability within one cell type. Despiteclonality, the Sca-1 outliers had distinct transcriptomes. Although theunique gene expression profiles eventually reversed to that of themedian cells, they lasted long enough to confer a greatly differentproclivity for choosing either the erythroid or myeloid lineages. Preference in lineage choice was associated with elevated expression oflineage-specific transcription factors, such as > 200-fold increase inGATA1 among the erythroid-prone cells, or > 15-fold PU.1 expressionamong myeloid-prone cell. Thus, clonal heterogeneity of gene expressionlevel is not due to independent noise in the expression of individualgenes, but reflects metastable states of a slowly fluctuatingtranscriptome that is distinct in individual cells and may govern thereversible, stochastic priming of multipotent progenitor cells in cellfate decision.
Keywords: murine EML cell line

overall_design: In the study presented here, the highest, middle, and lowest 15% Sca-1expressors from a clonal population of EML cells were used to acquireexpression profiles of a total of 46,628 unique genes using the IlluminaMouse-6 v1.1 microbead chips. Here, the Rank-Invariant normalized datais provided along with the individual raw data files.

middle: H

first: Sui

last: Huang


title: Gene expression profiling of overexpression of myocardin

status: Public on Jul 15 2008

city: Leiden

first: John

last: van Tuyn

address: Albinusdreef 2

department: Cardiology

institute: Leiden university Medical Center

summary:
ABSTRACT
Aims Forced differentiation of non-muscle cells into heart muscle cells using cardiac transcription factors (cTFs) may constitute a novel strategy to accomplish myocardial regeneration. Methods We investigated the potential of the cTF myocardin to induce cardiomyocyte differentiation and compared the myocardin-induced gene expression program to that of fully differentiated cardiomyocytes using oligonucleotide microarray hybridizations, quantitative RT-PCR analyses and immunofluorescence microscopy. The experiments were performed in the recently described human cardiomyocytes progenitor cells (hCMPCs), which differentiate into fully functional cardiomyocytes upon stimulation with 5-azacytidine and transforming growth factor β1. Results and Conclusions Forced myocardin expression stimulated transcription of a surprisingly large repertoire of heart muscle-specific genes in hCMPCs but did not cause their differentiation into functional cardiomyocytes. Specifically, myocardin gene transfer did not stimulate the synthesis of several sarcomeric (regulatory) proteins and ion channel constituents. The heart and smooth muscle-enriched isoforms of myocardin stimulate equally well the transcription of many of their cardiomyocyte-specific and virtually all of their smooth muscle target genes. However, the heart muscle-enriched myocardin species is a much more potent transactivator of a subset of genes encoding mainly cardiomyocyte-specific myofibrillar components. This may explain the underestimation of myocardin's cardiomyogenic potential in previous studies utilizing the smooth muscle-enriched isoform of this cTF.

overall_design:
Experiment performed on primary cells in culture, the cardiomyocyte progenitor cells (CMPCs), ScaI+ cells isolated from human fetal heart.

Cells tranduced with third gen lentivirus vectors, to allow stable overexpression of:
1.) CM-MYOCD cardiomyocyte specific isoform of human myocardin. n=4
2.) SM-MYOCD smooth muscle specific isoform of human myocardin (major open reading frame only). n=3
3.) SM-MYOCD smooth muscle specific isoform of human myocardin (both open reading frames). n=3
4.) LacZ transduction/vector/overexpression control. n=3
5.) untransduced. n=3

13 days post transduction, total RNA harvested for array.

first: Joost

middle: P

last: Sluijter

first: Jim

last: Swildens

first: Marie-José

last: Goumans

first: Pieter

last: Doevendans

first: Arnoud

last: van der Laarse

first: Martin

last: Schalij

first: Douwe

last: Atsma

first: Antoine

last: de Vries


title: Expression data from an involution time course in wild type and E2F3 +/- mice.

status: Public on Jul 11 2008

city: East Lansing

first: Eran

last: Andrechek

state: MI

address: 2120 BPS Building

department: Physiology

institute: Michigan State University

summary:
We have compared an involution timecourse for wild type and E2F3 heterozygous mice on the Operon 3.0 platform.  Three mice were taken for each genotype at each timepoint (Involution day 0, 1, 2, 3 and 4) and RNA was prepared from the mammary glands.  The RNA from each mouse was pooled and run as an individual sample.
Keywords: Mouse Mammary Gland RNA

overall_design: 5 timepoints, 2 genotypes, 3 replicates per timepoint.

first: Joseph

last: Nevins


title: Genomics of signalling crosstalk of estrogen receptor alpha in breast cancer cells

city: Oxford

first: Peter

last: Dudek

state: Oxfordshire

address: South Parks Road

country: United Kingdom

institute: University of Oxford

summary:
The estrogen receptor a (ERa) is a ligand-regulated transcription factor. However, a wide variety of other extracellular signals can activate ERa in the absence of estrogen. The impact of these alternate modes of activation on gene expression profiles has not been characterized. We show that estrogen, growth factors and cAMP elicit surprisingly distinct ERa-dependent transcriptional responses in human MCF7 breast cancer cells. In response to growth factors and cAMP, ERa primarily activates and represses genes, respectively. The combined treatments with the antiestrogen tamoxifen and cAMP or growth factors regulate yet other sets of genes. In many cases, tamoxifen is perverted to an agonist, potentially mimicking what is happening in certain tamoxifen-resistant breast tumors and emphasizing the importance of the cellular signaling environment. Using a computational analysis, we predicted that a Hox protein might be involved in mediating such combinatorial effects, and then confirmed it experimentally. Although both tamoxifen and cAMP block the proliferation of MCF7 cells, their combined application stimulates it, and this can be blocked with a dominant-negative Hox mutant. Thus, the activating signal dictates both target gene selection and regulation by ERa, and this has consequences on global gene expression patterns that may be relevant to understanding the progression of ERa-dependent carcinomas.
Keywords: treatment response

overall_design: 10 treatment conditions (3 biological replicates for each, totalling 30 individual samples), of which 3 were untreated controls. Each replicate was hybridized to a separate chip, totalling 27 cDNA slides representing 9 unique conditions.

first: Didier

last: Picard


title: Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors on NIA 15k microarray

first: Stéphane

last: LE CROM

summary:
Branchiomotor neurons are an important class of cranial motor neurons that innervate the branchial-arch-derived muscles of the face, jaw and neck. They arise in the ventralmost progenitor domain of the rhombencephalon characterized by expression of the homeodomain transcription factors Nkx2.2 and Phox2b. Phox2b in particular plays a key role in the specification of branchiomotor neurons. In its absence, generic neuronal differentiation is defective in the progenitor domain, and no branchiomotor neurons are produced. Conversely, ectopic expression of Phox2b in spinal regions of the neural tube promotes cell cycle exit and neuronal differentiation and at the same time induces genes and an axonal phenotype characteristic for branchiomotor neurons. How Phox2b exerts its pleiotropic functions, both as a proneural gene and a neuronal subtype determinant, has remained unknown.
To gain further insight into the genetic programme downstream of Phox2b we searched for novel Phox2b-regulated genes by cDNA microarray (here NIA 15k slides) analysis of facial branchiomotor neuron precursors from heterozygous and homozygous Phox2b mutant embryos.
Keywords: Phox2b-regulated genes identification

overall_design: Four biological replicates each in dye swap

first: Patrick

last: Pla

first: Stephane

last: Le Crom

first: Christo

last: Goridis


title: Lichen Planopilaris and Pseudopelade of Brocq Involve Distinct Disease Associated Gene Expression Patterns by Microarray

city: Vancouver

first: Kevin

last: McElwee

state: BC

department: Dermatology and Skin Science

institute: University of British Columbia

summary:
Lichen planopilaris (LPP) and pseudopelade of Brocq (PPB) are two scarring alopecia diagnoses that often exhibit similar clinical features. Some suggest LPP and PPB are not distinct diseases, but rather different clinical presentations in a spectrum derived from the same underlying pathogenetic mechanism. We explored the degree of similarity between LPP and PPB gene expression patterns and the potential for common and unique gene pathway and gene activity in LPP and PPB using microarrays.

Microarray analysis, using a 21K cDNA set was performed on pairs of biopsies obtained from affected and unaffected scalp of untreated patients (4 LPP and 4 PPB biopsy pairs). Diagnosis was confirmed by histopathology. Significantly differentially expressed genes were identified by analysis of microarray results in various data sets and screened for signaling pathway involvement. Selected genes were validated by quantitative PCR and immunohistology.

The global gene expression profiles in LPP and PPB versus comparative intra-control scalp tissue were distinguishable by Significance Analysis of Microarrays (SAM). Specific genes, such as MMP11, were identified with significantly differential expression in association with LPP versus PPB. There was very limited commonality in the gene expression profiles between LPP and PPB.

Our findings may have important implications for understanding the pathogenesis of LPP and PPB at the molecular level. Results suggest LPP and PPB involve different mechanisms of disease development and can be regarded as biologically distinct cicatricial alopecia diagnoses. Genes that we have identified may potentially be useful as markers of the respective diagnoses.

overall_design:
Human LPP and PPB biopsies were collected from study volunteers in the Department of Dermatology and Skin Science, University of British Columbia. Only patients that had not been previously treated with any therapeutic approaches were approached.
Patients were diagnosed with active phase LPP or PPB by clinical evaluation including a positive pull test when possible in those with LPP, and a possible pull test when possible in the case of PPB. Two tissue samples were then collected from each volunteer for laboratory analysis with one biopsy taken from the edge of an active lesion with perifollicular scale, with or without erythema, and the second biopsy from an unaffected region of scalp away from the lesional skin.
Human Operon v.2.1 (21K) glass arrays were produced (based on human 70mers from Operon Biotechnologies Inc, Huntsville, AL) by the Microarray Facility of the Prostate Centre at Vancouver General Hospital, Vancouver, Canada [17, 18]. RNAs were amplified using the SenseAmp Plus kit (Genisphere Inc, Hatfield, PA). The calculated A 260/280 ratio was used to determine the appropriate amount of sense RNA for labeling. Total RNA from test samples and universal human reference RNA (Stratagene, Cedar Creek, TX) were differentially labeled with Cy5 and Cy3 respectively, with the 3DNA array detection 350 kit (Genisphere Inc, Hatfield, PA) and cohybridized to cDNA microarrays. Following overnight hybridization and washing, arrays were imaged using a ScanArray Express scanner (PerkinElmer, Boston, MA).

first: Mei

last: Yu

first: Robert

last: Bell

first: Elizabeth

middle: K

last: Ross

first: Blanche

last: Lo

first: Megan

last: Isaac-Renton

first: Magda

last: Martinka

first: Anne

last: Haegert

first: Jerry

last: Shapiro


title: TEV-Arabidopsis pathosystem

status: Public on Jun 23 2008

city: Valencia

first: Miguel

last: Perez-Amador

state: Valencia

address: Avda. de los Naranjos s/n

institute: Inst. Biologia Molecular y Celular de Plantas (IBMCP-CSIC)

summary:
Transcription profiling of Arabidopsis plants infected with the Tobacco etch potyvirus TEV and the evolved TEV-At17, which was generated by serial passes of ancestral TEV in Arabidopsis. Infection with TEV-At17 cause severe symptoms in the plant, compared with the asymptomatic TEV-infection.
Keywords: Virus infection

overall_design: Five replicates for each sample category (Arabidopsis Ler infected with mock, or with TEV and TEV-At17 viruses) were generated, and compared with a global reference, generated from an equimolar mix of amplified RNAs from each of the 15 plants. Three replicas (#1,2, and 3) in each group were labeled with Cy5 and compared with Cy3-labeled reference mix, while the other two replicas (#4 and 5)  were reversed-labeled.

first: Pablo

last: Carbonell

first: Patricia

last: Agudelo-Romero

first: Santiago

middle: F

last: Elena


title: Performance comparison of Affymetrix and Illumina microarray technologies_IlluminaDataSet

status: Public on Jun 27 2008

city: Paris

first: Francois

last: Cambien

state: France

address: 91 Bd de l'Hôpital

department: Cardiovascular Genomics

institute: INSERM

summary:
The present study was conducted to compare the ability of Affymetrix and Illumina microarray technologies to characterize the differential gene expression profiles of human monocytes and monocyte-derived-macrophages.
Keywords: Cross-platform comparison

overall_design:
Blood samples were obtained from patients with symptoms of acute coronary syndrome who had undergone coronary angiography at the department of cardiology of the Pitié-Salpêtrière Hospital, Paris and who had one stenosis >50 % diagnosed in at least one major coronary artery.
RNA samples were extracted from monocyte and monocyte derived macrophage cells. Five biological replicates for each type of sample were hybridized in parallel to the Affymetrix HGU133 plus 2.0 and Illumina Bead Chip Human-6 v1 platforms. In addition, a reference list of differentially expressed genes was generated from a larger number of hybridizations (mRNA from 86 individuals) using a third technology (the academic RNG/MRC two-color chip) and was used as reference to assess the performance of the 2 platforms based on 10 samples. The internal consistency of each platform, the overlap in lists of genes identified as differentially expressed generated on each array type and the biological relevance of significant genes identified on each platform (Gene Ontology (GO) classes enrichments) were assessed.

first: Seraya

last: Maouche

first: Odette

last: Poirier

first: Tiphaine

last: Godefroy

last: Olaso

first: Ivo

last: Gut

first: Jean-Phillipe

last: Collet

first: Gilles

last: Montalescot

first: François


title: INDUCTION OF OXIDATIVE STRESS AND METAL BINDING GENES IN HUMAN ALVEOLAR MACROPHAGES EXPOSED TO FINE PARTICLES

status: Public on May 01 2008

city: Denver

first: Ivana

middle: V

last: Yang

state: CO

address: 1400 Jackson Street

institute: National Jewish Health

summary:
Exposure to particulate matter (PM) is consistently associated with increased morbidity and mortality attributable in part to respiratory illnesses. The alveolar macrophage (AM) is one of the cell types in the lung constantly exposed to and activated by ambient pollutants. Upon contact with environmental particulate pollutants, AM produces reactive oxygen species (ROS) and antioxidant enzymes, but the scope of this oxidative stress response induced by PM remains poorly defined. In this study, we used microarray analysis to determine the gene expression profile in human alveolar macrophages upon exposure to PM and sought to gain more insight into the global response of pro- and anti-oxidant enzymes to PM exposure. Human AM were obtained by bronchoscopy from normal individuals. They were then incubated with Chapel Hill PM2.5 (1 ug/ml) or vehicle for 4 hours (n = 6 independent samples each). mRNAs were extracted, amplified and hybridized to Agilent Human 1A microarray. Differentially expressed genes were identified by Statistical Analysis for Microarrays (SAM) with a FDR of 10% and a P ≤ 0.05. Significant genes were also mapped with Gene Ontology (GO) based on their molecular function. We found 34 and 41 up- and down-regulated genes respectively. Of these, 22 genes (~30%) were involved in metal binding and 14 genes were linked to oxidative stress, including 5 metallothionein-1 (MT1) isoform genes. In lung cells, addition of MT-1F in the medium attenuated PM2.5-induced H2O2 production while knockdown of MT1F gene expression increased H2O2 and IL-6 release induced by PM2.5. Our microarray experiments provided a global view of gene expression after in vitro PM2.5 exposure in human AM. The expression profile was most notable for differential expression of genes related to metal binding and oxidative stress, especially upregulation of MT1 isoform genes. Our findings suggest that metals associated with PM, e.g., zinc, copper, and arsenic, induce MT-1 and may be the primary mediators for PM-induced oxidative stress.
Keywords: in vitro exposure, PM2.5 particle treatment, human alveolar macrophages

overall_design: Total RNA was isolated from alveolar macrophages from 6 subjects were treated in vitro with PM2.5 particles or vehicle control for 4 hrs.  All particle-treated samples were co-hybridized with the common reference sample (pool of all 6 vehicle-treated samples) twice with Cy3 and Cy5 dyes flipped (12 hybridizations).  Three samples were also co-hybridized with its own individual control (vehicle-treatment for the same subject) with Cy3 and Cy5 dyes flipped (6 additional hybridizations).

first: Yuh-Chin

last: Huang

first: Zuowei

last: Li

first: Jackie

last: Carter

first: Joleen

last: Soukup

first: David

last: Schwartz


title: Microarray-based global mapping of integration sites of retrotransposon, intracisternal A-particle, in mouse genome

status: Public on May 03 2008

city: Chiba

first: Takashi

last: Takabatake

state: Chiba

address: Anagawa 4-9-1, Inage-ku

country: Japan

institute: National Institute of Radiological Sciences

summary:
Mammalian genomes have evolutionary harbored numerous mobile elements, a part of which are still active and evoke genomic instability.  Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression and disruption of neighboring genes in the host genome.  Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposons in mammalian genome can be identified at a time.  By this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA-integrating genomic regions.  Among 147 putative insertions located nearby the promoter regions, 91 were considered to be present discriminatively in the genome either of C3H/He or C57BL/6J mice, suggesting the existence of considerable strain differences.  In addition, by comparing genomic DNAs from a radiation-induced myeloid leukemia and its reference normal tissue, we detected 3 genomic regions around which an IAP element was integrated. These results demonstrate for the first time the successful genome-wide survey of a type of retrotransposon in mammalian genome.
Keywords: retrotransposon mapping

overall_design: Genomic DNA fragments containing sequences that flank a mouse retrotransposon are purified and hybridized to the microarray. Experiments are comparisions between signal intensities obtained from Cy3-labeled purified DNA fragments (test) and Cy5-labeled whole genomic DNA fragments (reference).

first: Hiroshi

last: Ishihara

first: Yasushi

last: Ohmachi

first: Izumi

last: Tanaka

first: Masako

middle: M

last: Nakamura

first: Katsuyoshi

last: Fujikawa

first: Tokuhisa

last: Hirouchi

first: Shizuko

last: Kakinuma

first: Yoshiya

last: Shimada

first: Yoichi

last: Oghiso

first: Kimio


title: The salivary transcriptome of Anopheles gambiae (Diptera: Culicidae) larvae: A microarrray-based study

city: St. Augustine

first: Marco

middle: V.

last: Neira

state: FL

address: 9505 Oceanshore Blvd.

department: The Whitney Laboratory

institute: University of Florida

summary:
In spite of the many recent developments in the field of vector sialomics, the salivary glands of larvalmosquitoes have been largely unexplored. We used whole-transcriptome microarray analysis to create a gene-expression profile of the salivary gland tissue of fourth-instar Anopheles gambiae larvae, and compare it to the gene-expression profile of a matching group of whole larvae. We identified a total of 221 probes with expression values that were (a) significantly enriched in the salivary glands, and (b)sufficiently annotated as to allow the prediction of the presence/absence of signal peptides in their corresponding gene products. Based on available annotation of the protein sequences associated with these probes, we propose that the main roles of larval salivary secretions include: (a) immune response, (b) mouthpart lubrication, (c) nutrient metabolism, and (d) xenobiotic detoxification. Other highlights of the study include the cloning of a transcript encoding a previously unknown salivary defensin (AgDef5), the confirmation of mucus secretion by the larval salivary glands, and the first report of salivary lipocalins in the Culicidae.
Keywords: Anopheles gambiae, salivary gland, Diptera, gene expression, salivary defensin, transcriptome, salivary lipocalin

overall_design: We attempted to identify transcripts that are both present and significantly enriched in the salivary glands (SG) of the 4th instar Anopheles gambiae larva. Transcripts were considered as present in the SG if the built-in Affymetrix detection call scored them as such in all three biological replicates of the experiment. Expression values (i.e. signal intensities) of transcripts from the salivary glands were compared to those of a matching group of whole 4th instar larvae using a t-test. Transcripts showing an expression value two-fold or higher (p < 0.01; false discovery rate=1%) in the SG as compared to whole larvae were considered to be significantly enriched.

last: Neira Oviedo

first: Jose

last: Ribeiro

first: Andreas

last: Heyland

first: Leslie

last: vanEkeris

first: Paul

last: Linser


title: Differentially expressed genes in radioresistant nasopharyngeal cancer cells

city: Teipei

first: Yun-Shien

last: Lee

address: No.199, Tunghwa Rd.,

institute: Chang Gung Memorial Hospital

summary:
The NPC parental cell lines were seeded at a density of 1 x 105/T25 flask (30% to 40% confluency) in complete medium. After 24 hours of cell seeding, various doses from 6 to 14 Gy were administered to determine the sublethal dose for each cell line. The radiation was delivered at room temperature at 300 cGy/min with a linear accelerator (2100EX, Varian, VA, USA). To establish RR subclone, the NPC cells were received the sublethal dose of irradiation. After treatment, the surviving cells were selected and cultured to produce first generation of the subline cells. Again, the subline cells were received sublethal dose of irradiation and the surviving cells were cultured to produce next generation of the subline cells. Up to four generations of NPC cells were produced and defined the forth generation cells as RR subclone.
Keywords: Parental vs. subclone cell line comparison

overall_design:
Array 1, NPC-076 Cy5 vers. NPC076-RR Cy3
Array 2, NPC-076-RR Cy5 vers. NPC-076 Cy3
Array 3, NPC-BM1 Cy5 vers. NPC-BM1-RR Cy3
Array 4, NPC-BM1-RR Cy5 vers. NPC-BM1 Cy3

first: JTC

last: Chang

first: SH

last: Chan

first: CY

last: Lin

first: HM

last: Wang

first: CT

last: Liao

first: TH

first: TY

first: AJ

last: Cheng


title: Investigation of gene expression signature induced by Alk5TD (active type I TGF-beta receptor)

status: Public on Apr 30 2008

city: Nashville

first: Shizhen Emily

state: TN

address: 2220 Pierce AVE, PRB-771

department: Cancer Biology

institute: Vanderbilt University

summary:
BT474 cells stably expressing Alk5TD or vector (BT474-Alk5TD and BT474-pBMN, respectively) were compared for gene expression. Genes that were differentially expressed between the two cell lines were collected as the signature induced by Alk5TD expression.
Keywords: Comparison of two cell types (BT474-Alk5TD and BT474-pBMN). BT474-pBMN was used as the reference line.

overall_design: RNA samples extracted from the two cell lines (BT474-Alk5TD and BT474-pBMN) were differentially labeled (BT474-Alk5TD with Cy5 and BT474-pBMN with Cy3) and analyzed. The experiment was performed in triplicate with independent samples.

first: Shizhen

middle: E

first: Carlos

last: Arteaga


title: Gene Expression Profiling of Single Embryos, Significant Effects of In Vitro Maturation, Fertilization and Culture

status: Public on May 05 2008

city: Storrs

first: Xiuchun

last: Tian

state: CT

address: 1392 Storrs Rd. Unit 4243

department: Center for Regenerative Biology

institute: University of Connecticut

summary:
	In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture, we compared the expression profiles of single bovine blastocysts generated by: 1) in vitro maturation, fertilization and culture (IVF); 2) in vivo maturation, fertilization and in vitro culture (IVD); and 3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367 and 200 genes differentially expressed between the AI and IVD, IVF and IVD and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category “RNA processing” was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on embryonic gene expression. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI to IVD embryos.
Keywords: developmental condition

overall_design: Reference design with dye swap. 43 individual embryos were analyzed.

first: Sadie

last: Smith

first: Robin

last: Everts

first: Li-Ying

last: Sung

first: Fuliang

last: Du

first: Raymond

last: Page

first: Boyd

last: Henderson

first: Sandra

last: Rodriguez-Zas

first: Tshimangadzo

last: Nedambale

first: Jean-Paul

last: Renard

first: Harris

middle: A

last: Lewin

first: Xiangzhong

last: Yang


title: Chimp 190 biopsy baseline vs post infection

status: Public on Apr 10 2008

city: rockville

first: Mike

last: havert

address: 1401 Rockville Pike

institute: CBER/FDA

summary:
Transcriptional profiling of chimp 190 liver biopsy comparing baseline sample with biopsy taken different days after innoculation with HCV
Keywords: Genetic modification

overall_design: Two-condition experiment, baseline vs. infected at weeks 19, 20, 21 and 32 post infection: One replicate per array.

last: Havert

first: Santosh

last: Nanda