title: Impact of normalization on Agilent miRNA microarray expression profiling

status: Public on Feb 17 2009

first: Sylvain

last: Pradervand

address: Genopode

institute: UNI Lausanne

summary:
Profiling miRNA levels in cells with miRNA-microarrays is becoming a widely used technique. Although normalization methods for mRNA gene expression arrays are well established, miRNA array normalization has so far not been investigated in detail. In this study we investigate the impact of normalization on data generated with the Agilent miRNA array platform. Here, we developed a method to select non-changing miRNAs (“invariants”) and used them to compute linear regression normalization coefficients or Variance Stabilizing Normalization (VSN) parameters. We compared the invariant normalizations to normalization by, scaling, quantile and VSN with default parameters as well as to no normalization using samples with strong differential expression of miRNAs (heart-brain comparison) and samples where only few miRNAs are affected (p53 overexpression in SCC13 cells versus GFP vector control transfected cells). All normalization methods performed better than no normalization. Normalizations procedures based on the set of invariants and quantile were the most robust over all experimental conditions tested. Our method of invariant selection and  normalization is not limited to Agilent miRNA arrays and can be applied to other datasets from one color miRNA microarray platforms, focused gene expression arrays and gene expression analysis using quantitative PCR.

Keywords: miRNA profiling

overall_design: To assay technical reproducibility, 3 technical replicates from brain and heart RNA (Stratagene) were hybridized on Agilent human miRNA microarrays. To determine sensitivity and specificity, heart and brain RNA were mixed in the following ratios: 50% heart 50% brain, 25% heart 75% brain and 5% heart 95% brain. Each of the dilutions was hybridized in a technical duplicate on Agilent human miRNA arrays. To assay the effect of p53 expression on miRNA levels in the human SCC13 cell line, RNA from 3 biological replicates of p53-expressing or control cells were hybridized in technical duplicates on the microarrays, resulting in a total of  12 hybridizations.

first: Keith

last: Harshmann

first: Johann

last: Weber

first: Jérôme

last: Thomas


title: Cancer Genome Anatomy Project (CGAP) Human Solexa LongSAGE

status: Public on Mar 20 2009

city: Bethesda

first: Cancer Genome Anatomy Project

last: CGAP

state: MD

department: Cancer Genome Anatomy Project

country: USA

institute: National Cancer Institute

summary:
This series represents the Cancer Genome Anatomy Project SAGE library collection.  Libraries contained herein were either produced through CGAP funding, or donated to CGAP.
The Cancer Genome Anatomy Project (CGAP: http://cgap.nci.nih.gov) is an interdisciplinary program established and administered by the National Cancer Institute (NCI: http://www.nci.nih.gov) to generate the information and technological tools needed to decipher the molecular anatomy of the cancer cell.

SAGE libraries are named according to the following convention:
* SAGE_Organ_histology_code_unique identifier, e.g., SAGE_Colon_adenocarcinoma_CL_Caco2
* Codes: B = bulk; CL = cell line; CS = short-term cell culture; MD = micro-dissected; AP = antibody purified.

overall_design: 36 libraries

first: R

middle: L

last: Strausberg

first: D

last: Gerhard

first: Gregory

middle: J

last: Riggins


title: Notch targets in KC Drosophila cells

city: Cambridge

first: Sarah

last: Bray

address: Downing Street

department: PDN

country: United Kingdom

institute: University  of Cambridge

summary:
To identify genes upregulated in response to Notch signalling in KC cells.
Keywords: Expression analysis at a single timepoint (30' after Notch activation)

overall_design: KC cells were obtained from Dr Martin Zeidler. Control versus Notch activated KC cells: mRNA was extracted from KC cells incubated in the absence or presence of EDTA to activate Notch (cells were harvested 30 minutes after addition of EDTA). RNA was isolated using TRIzol (Sigma) and reverse transcription was performed with Superscript III Reverse Transcriptase (Invitrogen) and oligo-dT primers (Sigma). Control and experimental samples were labeled with Cy3 or Cy5, mixed together and hybridized on Drosophila transcriptome long-oligonucleotide microarrays (FlyChip, FL002;  http://www.flychip.org.uk/services/core/FL002/). Three biological replicates and dye swaps were performed (5 arrays in total/experiment). Slides were scanned by Genepix 400B dual laser scanner (Axon) and spots were found and quantified by Dapple software.

first: Alena

last: Krejci


title: Mismatch oligonucleotides in human

status: Public on Feb 18 2009

city: New Haven, BASS 437

first: Michael

middle: R

last: Seringhaus

state: CT

address: 266 Whitney Ave

department: Molecular Biophysics & Biochemistry

institute: Yale University

type: Genome variation profiling by genome tiling array

summary:
Tiled regions surrounding 5 human genes as 36mers, HBG2, TIMP3, SYN3, FLNA, FBX07. The first three of these genes, we tiled with various mismatch oligos in addition to 'perfect match' oligos.

Keywords: Mismatch hybridization experiment

overall_design: Tiled perfect match and various designs of mismatch oligonucleotide for several human genes. Goal was to observe the influence of various MM types on hybridization behavior in human, and compare it to yeast (see related slide).

first: Joel

last: Rozowsky

first: Thomas

last: Royce

first: Ugrappa

last: Nagalakshmi

first: Justin

last: Jee

last: Snyder

first: Mark

last: Gerstein


title: Gene expression profiling via LongSAGE in a non-model plant species: A case study in seeds of Brassica napus

status: Public on Feb 16 2009

city: Giessen

first: Christian

last: Obermeier

address: Heinrich-Buff-Ring 26-32

department: Plant Breeding

country: Germany

institute: Justus Liebig University

summary: Serial Analysis of Gene Expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana. Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression in about 10 % of all matched genes, with a total abundance of 44 %, showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6 % of A. thaliana genes were matched by Brassica ESTs detected by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development.

overall_design:
Seeds from 2 developmental stages of B. napus were used to construct 2 LongSAGE libraries, 23 days after pollination (23 DAP) and 35 days after pollination (35 DAP).
Biological replicates and confirmation: Cloning of tag-amplified RT-PCR products, Real-time RT-PCR

first: Bashir

last: Hosseini

first: Wolfgang

last: Friedt

first: Rod

last: Snowdon


title: microRNA-125b Promotes Neuronal Differentiation in Human Cells by Repressing Multiple Targets

status: Public on Feb 12 2009

city: Singapore

first: Haw Siang

last: Ang

address: 8A Biomedical Grove

country: Singapore

institute: SIgN

summary:
We report here the expression profile of microRNAs in human neuronal differentiation in the neuroblastoma cell line SH-SY5Y. Six microRNAs were significantly upregulated during differentiation induced by all-trans¬-retinoic acid and brain-derived neurotrophic factor. We demonstrated that ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both spontaneous and induced differentiation of SH-SH5Y cells, based on neurite outgrowth and neuronal marker expression. In human neural progenitor ReNcell VM cells, miR-125b is also upregulated during differentiation and miR-125b ectopic expression significantly promotes neurite outgrowth. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the microRNA and/or predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonize neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the microRNA response elements of ten selected targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation.

Keywords: mir125-OE/mir-scrambled control  comparison & mir125-KD/mir-scrambled control comparsion

overall_design: In this study we sought to understand the role of miRNAs in differentiation of human neural cells using simple in vitro models, human neuroblastoma SH-SY5Y cells and human neural progenitor ReNcell VM cells. When sequentially treated with all-trans-retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), SH-SY5Y cells give rise to fully differentiated neuron-like cells (8). These differentiated SH-SY5Y cells are withdrawn from the cell cycle, express various neuronal markers, and exhibit carbachol-evoked noradrenaline release (8). Moreover, as no glial cell is derived by this process, it is a robust and homogenous model system for investigating neuronal differentiation (8). Using microarrays and Northern blots, we identified a group of miRNAs that are significantly upregulated in differentiated SH-SY5Y cells. We further showed that one of these miRNAs, miR-125b, significantly enhances differentiation and neuronal morphogenesis of SH-SY5Y cells. In addition, this miRNA also promotes neurite outgrowth in human neural progenitor ReNcell VM cells.

first: Minh

last: Le

first: Huangming

last: Xie

first: Beiyan

last: Zhou

first: Poh

last: Hui Chia

first: Mookyoung

last: Um

first: Leonard

last: Lipovich

first: Gerald

last: Udolph

first: Henry

last: Yang

first: Bing

last: Lim

first: Harvey

last: Lodish


title: Effects of 35 days oral exposure of adult female Sprague-Dawley rats to p-tert-octylphenol on uterine gene expression

status: Public on Feb 20 2009

city: Laval

first: Patrick

last: Devine

state: QC

address: 531 boulevard des Prairies

department: INRS-Institut Armand-Frappier

country: Canada

institute: Institut national de la recherche scientifique

summary:
Contamination of the environment with endocrine disrupting chemicals (EDCs) has raised concerns about potential health hazards for humans and wildlife. P-tert-octylphenol (OP) is one such ubiquitous chemical reported to bind to the estrogen receptor (ER) and alter expression of estrogen-responsive genes. Human and wildlife exposure to OP are likely, due to its persistence in the environment and its presence in food, water and items of daily use. Detrimental effects of OP exposures on the reproductive system have been observed in some, but not all, in vivo experiments. This study compared estrogenic effects of OP in vitro with those in vivo in adult female rats, attempting to better mimic real-life exposures in adults. In vitro, OP bound to human ER weakly and accelerated proliferation of MCF-7 cells.  Adult Sprague-Dawley rats were given OP by gavage either once (125 mg/kg OP) or daily for 35 days (25, 50, 125 mg/kg).  Body and organ weights and ovarian follicle populations were unaltered in OP-exposed adult rats after either time point, despite detectable levels of OP in reproductive organs. Toxicity of OP was demonstrated by a slightly reduced growth rate and slightly altered estrous cycle, but there were no significant estrogen-like changes in histomorphology or microarray analyses of gene expression of uterine tissue.  Prepubertal rats exposed by gavage to 125 or 250 mg/kg OP for three days failed to show any uterotrophic effects, although E2 caused a 3-fold increase in uterine weight. These results do not support a dose-dependent, estrogenic mode of action for OP.

Keywords: treatment vs control

overall_design: Individual uterine RNA samples from 4 female 35-day old rats per treatment (4 OP and 4 vehicle) sacrificed on the second day of diestrus after 35 days of exposure to OP (125 mg/kg/day in propylene glycol) or solvent (propylene glycol, PG) by gavage were compared with liver RNA pooled from all 8 rats (as reference, combined OP and solvent treatment groups). Comparisons were made between treatments.  Liver cDNA was always marked with Cy5 and uterine cDNA was always marked with Cy3.

first: Sukhdeep

middle: K

last: Sahambi


title: Changes in gene expression in MKK2 and MKKKC5 mutants after Alternaria infection

status: Public on Feb 15 2009

city: BEAUCOUZE

first: Sandra

last: Pelletier

address: 42, rue Georges Morel - BP 60057

country: France

institute: INRA

summary:
The mkkkC5 mutant, a knockout line of MAPKKKC5, shows changes in growth and development, as well as an altered Alternaria sensitivity. It was shown by Brader and Djamei et al.(2007) that Alternaria brassicicola sensitivity is also influenced by MKK2-activity. Aim of this experiment is to access the transcriptional changes in the mkkkc5plants as well as in plants overexpressing a constitutively active MKK2-version (MKK2EE). The second part of the experiment addresses transcriptional changes in these plants after Alternaria brassicicola infection.
au07-09_alternaria
Keywords: treatment variations

overall_design:
Alternaria brassicicola infection of 25 day old mkkkc5- , pGreen::MKK2EE-myc and WT Col-0 plants. Sampling after 0, 1 and 2 days. Comparison between mutants and WT at corresponding timepoints. Additionally comparison between WT-samples at different timepoints.
16 dye-swap - CATMA arrays

first: Jean-Pierre

middle: P

last: Renou

first: Heribert

last: Hirt

first: Sarah-

last: Himbert

first: Marie-Laure

last: Martin-Magniette


title: Legionella pneumophila JR32 and rpoS- (LM1367) strains: Statinoary phase (stat) vs. Logarithmic (log) phase growth

status: Public on Feb 14 2009

city: new york

first: Galadriel

middle: A

last: Hovel-Miner

state: NY

department: Microbiology

institute: Columbia University

summary:
Transcriptional profiling of L. pneumophila JR32 (wild-type) and rpoS- (LM1376) strains grown to stat phase compared to cultures grown to log phase

Keywords: growth phase

platform_organism: Legionella pneumophila subsp. pneumophila str. Philadelphia 1

overall_design: For each of the two strains, six test samples and six reference samples were analyzed and averaged. The test sample being JR32 or rpoS- in stat phase and the reference sample being JR32 or rpoS- in log phase.

first: Sergey

last: Pampou

first: Sebastien

last: Faucher

first: Margaret

last: Clarke

first: Irina

last: Morozova

first: James

last: Russo

last: Kalachikov

first: Howard

last: Shuman


title: StaphVar-DNA microarray analysis of accessory genome of community-acquired methicillin resistant Staphylococcus aureus

city: Brussels

first: Ricardo

middle: Luis

last: De Mendonca

address: Route de Lennik 808

country: Belgium

institute: ULB - Hôpital Erasme

summary: Approximatively 75% of the genome of S. aureus (“core” genome) are highly conserved between strains, whereas the remaining 25% (“accessory” genome) are composed of variable regions that are mostly composed of mobile genetic elements (MGE), containing virulence and resistance genes. We have developed a composite DNA-microarray (StaphVar Array) which selectively targets 403 genes located on the accessory or core variable genome. Target genes encode antimicrobial resistance factors (35 %), virulence factors (28 %) and adhesins (31%). This microarray was validated with reference strains and used to characterize the genomic DNA of 13 community-acquired methicillin resistant S. aureus (CA-MRSA) strains representative of all the Multilocus Sequence Types (STs) described to date in Belgium. Analysis of gene content of 8 reference strains by the StaphVar Array matched 96 to 99% of the theoretical results. Analysis of CA-MRSA strains showed that 54.4 % of the genes tested were strain-dependent. Strains presented specific exotoxin, enterotoxin, cytolysin and adhesin gene profile by multi-locus sequence typing (MLST) lineage. One exception to these “lineage-specific” gene profile was the variable presence of the Arginin Catabolic Mobile Element (ACME, characteristic of the USA300 clone) within ST8 strains. In conclusion, this novel StaphVar array enables characterization of more than 400 variable resistance and virulence determinants in S. aureus strains. CA-MRSA strains from Belgium displayed extensive diversity in virulence and resistance profile. The presence of the USA 300 clone in our country was confirmed. Although mainly located on MGE, association of virulence genes were highly conserved within strains of the same MLST lineage.

overall_design: CGH microarray was performed on 13 epidemiologically distinct clinical isolates of methicillin resistant S. aureus. S. aureus labeled genomic DNA were hybridized to StaphVar arrays containing 1326 60mer oligonucleotide probes (Eurogentec, Belgium).

first: Farid

last: El Garch

first: Marie

last: Hallin

first: Anne

last: Lefort


title: Comparative Placental Efficiency in Swine Examined Using Expression Profiling

status: Public on Feb 06 2009

city: Raleigh

first: Nicholas

middle: Eldon

last: Hardison

state: NC

address: 4700 Hillsborough St., Room 318A

institute: North Carolina State University

summary:
Background: Genome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to identify significant breed-by-probe interactions.   Results:  Gene specific linear mixed models were fit to each of the log2 transformed probe intensities on these arrays, using fixed effects for breed, probe, breed-by-probe interaction, and a random effect for array.  After surveying the day 25 placental transcriptome, 789 probes with a q-value ≤ 0.05 and |fold change| ≥ 2 for the breed-by-probe interaction were identified as candidates containing SFP. To address the quality of the bioinformatics approach, universal pyrosequencing assays were designed from Affymetrix exemplar sequences to independently assess polymorphisms within a subset of probes. Of those probes sampled from high-, medium-, and low-ranking categories, 20 of 27 were confirmed by pyrosequencing to contain SFPs.  In most cases, the 25-mer probe sequence printed on the microarray diverged from Meishan, not occidental crosses. This analysis was used to define a set of highly reliable predicted SFPs according to their probability scores.   Conclusions:  By this method we detected transition and transversion single nucleotide polymorphisms, as well as insertions/deletions.  These results demonstrate that this approach can identify polymorphisms between two breeds and/or lines of any species for which a short oligonucleotide array is available, and can be used to rapidly develop markers for genetic mapping and association analysis in species where high density genotyping platforms are otherwise unavailable.  SNPs and INDELS discovered by this approach have been publicly deposited in NCBI’s SNP repository dbSNP. This method is an attractive bioinformatics tool for uncovering breed-by-probe interactions, for rapidly identifying expressed SNPs, for investigating potential functional correlations between gene expression and breed polymorphisms, and is robust enough to be used on any Affymetrix gene expression platform.
Keywords: Transcriptional profiling of Day 25 porcine placentas

overall_design: 30 samples: Breed (Meishan, Occidental) Placental Tissue, Days (25, 45, 65, 85, 105)

first: Steve

last: Bischoff

first: Shengdar

last: Tsai

first: Jorge

last: Piedrahita


title: Tissue-Specific Transcriptional Profiling of Epigenetic Asymmetry in Porcine Parthenogenetic Fetuses1

summary:
Using two complementary approaches, analysis of imprinting of candidate genes by pyrosequencing and expression profiling of parthenogenetic fetuses, we carried a comprehensive survey of genomic imprinting in swine. In the case of imprinted genes where transcription of one of the two parental alleles is silenced, uniparental embryos like parthenotes can be used to measure transcript dosage effects. Using Affymetrix Porcine GeneChip microarrays, four tissues of day 30 fetuses were profiled: brain, fibroblast, liver, and placenta.
Keywords: Transcriptional profiling of epigenetic asymmetry in porcine day 30 parthenogenetic and biparental fetal tissues

overall_design: 24 samples total of Day 30 occidental porcine fetuses: 2 treatments (parthenote, control); four tissues (brain, fibroblast, liver, placenta); technical replicates (3 fibroblast only)


title: Effects of high concentrations of progesterone and Mifepristone: a  gene expression study of endometrial cancer cells

status: Public on Feb 05 2009

city: Tromsø

first: Ruth

middle: Hracky

last: Paulssen

address: MH-Bygget Breivika

department: Institute of Clinical Medicine

country: Norway

institute: Universitetet i Tromsø

summary: Patients with endometrial hyperplasia representing preliminary stages of endometrial cancer have shown to respond to therapy in 100% of the cases when treated with levonorgestrel -impregnated intrauterine device. Antiproliferative effect has also been reported after application of an anti-progestin impregnated intrauterine device which showed to induce endometrial atrophy. The intention of the present study was to obtain more information of novel therapeutic targets for hormonal treatment in endometrial hyperplasia and endometrial cancers. Gene expression of signaling pathways after stimulation of Ishikawa cells with high doses of progesterone (10 μM) or Mifepristone (10 μM) was performed. After using an oligo microarrays representing 24,650 human genes and 37,580 gene transcripts, 6,154 genes remained after pre-processing and filtering. This resulted in a total of 993 up-regulated genes with 189 genes for progesterone and 255 genes for Mifepristone. The 550 down regulated genes were distributed with 256 genes for progesterone, 127 genes for RU 486. The results showed that genes presenting the epidermal growth factor (EGF)/MAP-kinase pathway were significantly over-represented by progesterone treatment, whereas, by Mifepristone treatment genes involved in the p53 pathway were also up-regulated (data not shown). These genes may be interesting as potential new therapeutic targets in endometrial hyperplasia and endometrial cancer, as candidate genes for therapy response or as candidate markers for tumor progression.

overall_design:
20 samples, 4 different condition groups with 5 replicates for each group, linear factorial experimental design.
Short time (4hrs) dose response to progesterone and Mifepristone in combination and alone.
Only 18 samples are represented in this Series.

middle: H

last: Ørbo

first: Bjørn

middle: T

last: Moe

first: Halvor

last: Grønaas


title: FFPE study using MIP copy number platform - liver

city: Santa Clara

first: Yuker

last: Wang

state: CA

address: 3380 Central Expressway

institute: Affymetrix

summary:
:         A major challenge to the study of tumor DNA copy number (CN) in clinical specimens is the lack of appropriate fresh frozen samples and thus a dependence on Formalin-Fixed Paraffin Embedded (FFPE) banked samples, which typically have more extensive clinical follow up information. However, on most high density CN platforms, DNA from FFPE tissues generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from DNA isolated from cell lines and frozen tumor samples. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess FFPE samples. In this study, we successfully applied MIP technology with a panel of 50,000 markers to CN determination in FFPE samples. Using an input of 37 ng genomic DNA, we demonstrated high quali
 ty CN data with MIP technology from 93 FFPE samples from seven diverse collections. We found that the performance of FFPE DNA for CN determination was comparable to that of DNA obtained from matched frozen tumor, with only a modest loss in performance of DNA.

overall_design: Various collections of FFPE samples have been assayed by MIP

first: Victoria

last: Carlton

first: Malek

last: Faham


title: Gene expression profiles of progestin-induced canine mammary hyperplasia and spontaneous mammary tumors

status: Public on Feb 09 2009

city: Utrecht

first: Nagesha

middle: A S

last: Rao

state: Utrecht

department: Veterinary sciences

country: Netherlands

institute: Utrecht University

summary:
Spontaneous mammary tumours are the most prevalent type of neoplasm in women as well as in female dogs. Even though ovarian hormones, mainly estrogens and progestins, are known to play a key role in mammary tumorigenesis, conflicting reports have been obtained from in vivo and in vitro studies concerning their role, especially of progestins, in mammary tumorigenesis. Prolonged exposure to high levels of progestins during unusually long luteal phase of oestrous cycle is suspected to be the key event in canine mammary tumorigenesis.  Accordingly, our previous studies have shown development of mammary hyperplasia in dogs upon prolonged progestin administration. In this study, we used canine cDNA microarrays to identify oncogenic determinants in progestin-induced canine hyperplasia (CMH) and spontaneous tumours (CMC) by comparing them to normal tissue. CMH profile indicated amplification of genes involved in cell proliferation, like PCNA, NPY and RAN, and also alterations in expression of transcription factors and cell adhesion molecules. In addition to amplified cell proliferation signature, CMC profile also identified major alterations in expressions of genes involved in cell motility, cytoskeletal organization and extracellular components. Overall, gene expression profile of CMH indicated an advantage for cell proliferation, whereas that of CMC indicated an advantage for proliferation as well as neoclassic transformation.  In conclusion, our findings indicate strong oncogenic potential of progestins in canine mammary gland. In addition, CMC gene expression profile greatly contribute to the identification of their molecular abnormalities and may facilitate the identification of new therapeutic targets.

Keywords: Expression profiling by using cDNA microarray

overall_design: Gene expression changes in three different canine mammary tissues types, namely normal, progesterone-induced hyperplasia (CMH) and spontaneous tumors (CMC), were analyzed by comparing to a common reference. Total RNA was isolated from the canine mammary tissues used. In vitro transcription followed by labeling and hybridization to cDNA microarray was carried out. In a reference design of hybridization, labeled cRNA from each sample was hybridized against reference pooled cRNA. Out of 37 tissues samples used in this study, 8 were normal, 8 were CMH and 21 were CMC. Equal numbers of samples per tissue group were labeled with Cy3 and Cy5 (dye swap). Reference cRNA was prepared by adding an equal quantity of cRNA derived from individual samples used in this study. Statistical analysis of the log-transformed normalized data was done using SAM (significance analysis of microarray) and differentially expressed genes in CMH and CMC compared to normal tissue were identified.

first: Monique

middle: E

last: van Wolferen

first: Ana

last: Gracanin

first: Sofie

middle: F

last: Bhatti

first: Magdalena

last: Krol

first: Frank

middle: C

last: Holstege

first: Jan

last: Mol


title: Gene Expression Profiling of Tumor Microenvironment during Breast Cancer Progression

status: Public on Feb 04 2009

city: San Diego

first: Xiao-Jun

last: Ma

address: 11025 Roselle St, Suite 200

institute: bioTheranostics, Inc.

summary:
To better understand the role of tumor microenvironment in breast cancer progression, we combined laser capture microdissection and microarray analysis to provide a comprehensive catalog of gene expression changes in both tumor and tumor-associated stroma.

Keywords: cancer vs. normal

overall_design: We used LCM to isolate the epithelial and stroma compartments separately from each of 14 fresh frozen primary breast cancer biopsies. In the epithelial compartment, we captured normal (N) and malignant (DCIS or IDC or both where available) epithelium from each tissue slide. In the stroma compartment, we captured both normal stroma away from the malignant lesion (NSS) and the DCIS-associated stroma (ISS) and/or IDC-associated stroma (INVS) whenever possible.

first: Dennis

last: Sgroi


status: Public on Feb 01 2009

city: Boston

first: Julja

last: Burchard

state: MA

address: 33 Avenue Louis Pasteur

department: Computational & Systems Biology

institute: Merck & Co., Inc.

type:
Non-coding RNA profiling by array
Expression profiling by array

summary: siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand ‘‘seed region,’’ similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
Hepa1-6 mouse hepatoma cell line and HUH7 and PLC/PRF/5 human hepatoma cell lines were transfected in 6-well plates using Lipofectamine
RNAiMAX and siRNA duplexes at a final concentration of 10 nM. For in vitro analysis, RNA was extracted at 6, 12, 24, and 48 h post-transfection. For in vivo studies, mouse livers were harvested 3 d following a single administration of APOB siRNA (3 mg/kg) formulated in lipid nanoparticles.  For details, please see: J. Burchard, A.L. Jackson, V. Malkov, R.H.V. Needham, Y. Tan, S.R. Bartz, H. Dai, A.B. Sachs and P.S. Linsley.  microRNA-like off-target transcript regulation by siRNAs is species specific.  RNA 15 (2009)

first: Aimee

last: Jackson

first: Vladislav

last: Malkov

first: Rachel

last: Needham

first: Yejun

last: Tan

first: Steven

last: Bartz

first: Hongyue

last: Dai

first: Alan

middle: B

last: Sachs

first: Peter

middle: S

last: Linsley


title: High density Tiling array analysis of Streptococcus pneumoniae TIGR4 genome

status: Public on Feb 10 2009

city: starkville

first: Ranjit

last: Kumar

state: MS

address: 240 Wise Center Drive,PO Box 6100

department: Dept of Basic Science

institute: MSU

type: Expression profiling by genome tiling array

summary: The objective of the study is to obtain a high resolution transcription map of S. pneumoniae genome and make it available through a genome browser. The study also help to identify gene expression pattern, presence of small RNAs, Antisense expression and operon structure.

overall_design:
S. pneumoniae strain TIGR4 (ref) was grown in THYmedium, Todd-Hewitt broth supplemented with 0.5% yeast extract. Cells were harvested from duplicate cultures from mid-log phase (OD600 nm, 0.4–0.6) of growth by centrifugation. The harvested pellets were washed twice in sterile PBS (pH 7.0) and stored at -80°C. RNA was purified from frozen bacterial pellets using Qiagen RNeasy kit by following the manufacturer’s protocol. Isolated RNA was treated with DNase to remove genomic DNA contamination, and purity was checked by performing a one-step RT-PCR using primers specific for 16S rRNA in presence or absence of reverse transcriptase. RNA concentration and quality were determined by using Agilent Bioanalyzer. Purified RNA was stored in nuclease free water at -80°C. One microgram of total RNA was used by Nimblegen systems for labeling and hybridization.
2 biological replicates (single colored) were used in the analysis. 20,000 random probes were designed on the chip as a negative controls to measure non-specific hybridization. 35 genes identified through proteomics of same sample were used as positive controls.

first: Pratik

last: shah

first: Ed

last: Swiatlo

first: Mark

middle: L

last: Lawrence

first: Bindu

last: Nanduri

first: Shane

last: burgess


title: Small RNAs in rice seed

status: Public on Feb 02 2009

city: Shanghai

first: Hong-Wei

last: Xue

address: 300 Fenglin Road

country: China

institute: Institute of Plant Physiology and Ecology,Shanghai Institutes of Biological Sciences

summary: Small RNAs are common and effective modulators of gene expression in eukaryotic organisms. To characterize the small RNAs expressed during rice seed development, massively parallel signature sequencing (MPSS) was performed, resulting in the obtainment of 22-nt sequence signatures. Through integrative analysis,novel miRNAs were identified mostly based on the miRNA* accumulation.

overall_design: Total RNA was isolated separately from rice seeds collected at 3, 6, 9 and 12 days after anthesis (DAA), then mixed into a library and separated on denatured polyacrylamide gel electrophoresis (PAGE). The fraction of 18-26 nt small RNA was recovered by small RNA gel extraction Kit.

first: Liang-Jiao

first: Jing-Jing

last: Zhang


title: Transcriptome analysis of potato tubers: Effects of different agricultural practices

city: Wageningen

first: Jeroen

last: van Dijk

address: Bornsesteeg 45

institute: RIKILT, institute of food safety

summary:
The use of profiling techniques such as transcriptomics, proteomics, and metabolomics has been proposed to improve the detection of side effects of plant breeding processes. This paper describes the construction of a food safety-oriented potato cDNA microarray (FSPM). Microarray analysis was performed on a well-defined set of tuber samples of two different potato varieties, grown under different, well-recorded environmental conditions. Data were analyzed to assess the potential of transcriptomics to detect differences in gene expression due to genetic differences or environmental conditions. The most pronounced differences were found between the varieties Sante and Lady Balfour, whereas differences due to growth conditions were less significant. Transcriptomics results were confirmed by quantitative PCR. Furthermore, the bandwidth of natural variation of gene expression was explored to facilitate biological and/or toxicological evaluation in future assessments.

Keywords: experiment with factorial design

platform_organism:
Solanum tuberosum
Solanum phureja

overall_design: factorial design; 2 potato cultivars (Sante, Lady Balfour); 2 fertilizers (dairy manure compost, chicken manure pellets); 3 plant protection treatments (copper oxychloride, comcat, water), 3 biological replicates, 48 samples

first: Kok

middle: J

last: Esther

first: Katarina

last: Cankar


title: Woman skeletal muscle transcriptome with bed rest and countermeasures.

city: OTTAWA

first: Angele

last: CHOPARD

state: ONTARIO

address: 451 Smyth road

department: Faculty of Medicine

institute: University of Ottawa

summary: Microgravity has a dramatic impact on human physiology, illustrated in particular with skeletal muscle impairment. A thorough understanding of the mechanisms leading to loss of muscle mass and structural disorders is necessary for the definition of efficient clinical and spaceflight countermeasures. We investigated the effects of long-term bed rest on transcriptome of soleus (SOL) and vastus lateralis (VL) muscles in healthy women (BRC group, n=8), and the potential beneficial impact of protein supplementation (BRN group, n=8) and of a combined resistance and aerobic training (BRE group, n=8). Gene expression profiles were obtained using an in-house made microarray containing 6681 muscles-relevant genes. A two-class statistical analysis was applied on the 2103 genes with consolidated expression. We identified 472 and 207 modified genes, respectively for SOL and VL in BRC group.  Further clustering approaches, identifying relevant biological mechanisms or pathways, underlined five main subclusters. Three are composed almost of upregulated genes involved mainly in nucleic acid and protein metabolism, and two composed almost of downregulated genes involved in energy metabolism. Exercise countermeasure demonstrated a drastic compensatory effect, decreasing the number of differentially-expressed genes by 89 and 96% in SOL and VL. In contrast, nutrition countermeasure had a moderate effect and decreased the number of differentially-expressed genes by 40 and 25% in SOL and VL. Our results allowed reporting a systematic, global and comprehensive view of long-term woman muscle atrophy and brought new lights and insights for space environment and for women who undergo a long-term clinical bed rest.

overall_design: Biological samples were collected from Pre- and Post- bed rest (BR) soleus and vastus lateralis biopsies of each subject from the three groups (bed rest only: BRC; Exercise: BRE; Nutrition: BRN). six technical replicate values (2 duplicate hybridizations “a et b” to chips with triplicate spots “xxx”) were obtained for each skeletal muscle sample. Thus for each subject, 12 expression measurements (6 before BR and 6 after BR) were obtained for each muscle.

last: Chopard

first: Martine

last: Lecunff

first: Richard

last: Danger

first: Guillaume

last: Lamirault

first: Audrey

last: Bihouee

first: Raluca

last: Teusan

first: Bernard

last: Jasmin

first: Jean-Francois

last: Marini

first: Jean

last: Leger


title: Identification of Embryonic Pancreatic Genes using Xenopus DNA Microarrays

status: Public on Feb 03 2009

city: Chiyoda-ku

first: TADAYOSHI

last: HAYATA

address: 2-3-10 Kandasurugadai

department: Medical Research Institute

country: Japan

institute: Tokyo Medical and Dental University

summary:
The pancreas is an exocrine and endocrine endodermal organ involved in digestion and glucose homeostasis.  During embryogenesis, the anlagen of the pancreas arise from dorsal and ventral evaginations of the foregut that later fuse to form a single organ.  In order to better understand the molecular genetics of early pancreas development, we sought to isolate markers that are uniquely expressed in this tissue.  Microarray analysis was performed comparing dissected pancreatic buds, liver buds, and the stomach region of tadpole stage Xenopus embryos.  A total of 912 genes were found to be differentially expressed between these organs during early stages of organogenesis.  K-means clustering analysis predicted 120 of these genes to be specifically enriched in the pancreas. Of these, we report on the novel expression patterns of 24 genes.  Among them, STXBP6 and KIRREL2 were expressed by the early tailbud stage, suggesting the possible involvement of novel signaling pathways in early pancreas development.
Keywords: organ type comparison

overall_design: We wished to identify genes that are specifically expressed in the developing pancreas of early Xenopus embryos to better understand the process of endodermal organ differentiation.  During embryonic gut formation, the liver and pancreatic rudiments bud out from the epithelium of the developing digestive tube posterior to the future stomach (Kelly and Melton 2000; Chalmers and Slack, 1998).  The dorsal pancreatic bud originates from the gut tube underneath a portion of the notochord and the ventral pancreatic buds derive from the ventral gut adjacent to the heart.  These pancreatic primordia are independently induced and later fuse to form a single pancreas.  We isolated the pancreas, liver, and stomach buds from stage 42 embryos (swimming tadpole stage after the dorsal and ventral pancreatic buds fuse), and performed Affymetrix gene expression analyses using Xenopus laevis genome arrays (version 1.0).  These organ buds consist of the endoderm component, as well as also the endodermal and a mesodermal component.  Experiments were performed twice independently using two independently isolated tissue samples.  We used Expressionist software (GeneData) to refine microarray data and performed gene expression profiling analyses.  In order to identify the genes that are differentially expressed between the pancreas, liver, and stomach, we first selected genes that survived the analysis of variance (ANOVA) with p values of less than 0.05.  Four thousand two hundred two genes satisfied this criterion. Next, the expression levels of each gene in the pancreas, liver and stomach, were compared.  Genes with average expression difference ratios of greater than three-fold were selected for further analyses.  After manually removing genes that are redundantly represented in the Affymetrix chips, 912 genes survived the analyses.  These genes were separated into six expression groups based on K-means clustering analysis.  Group P represents 120 genes that are specifically enriched in the pancreas.  Group L represents 237 genes preferentially expressed in the liver, and 303 genes in Group S are enriched in the stomach.  Groups PL, PS and LS represent the genes that are enriched in both the pancreas and liver (90 genes), pancreas and stomach (88 genes), and liver and stomach (74 genes), respectively.

first: Tadayoshi

last: Hayata

first: Ira

last: Blitz

first: Nahoko

last: Iwata

first: Ken

middle: W

last: Cho


title: Increased physical activity accompanies carbohydrate preference in a B6.CAST-17 sub-congenic mouse strain

city: Baton Rouge

first: Brenda

last: Richards

state: LA

address: 6400 Perkins Rd.

department: Basic Science

institute: Pennington Biomedical Research Center

summary:
A C57BL6/J (B6) x CAST/Ei (CAST) strain intercross was used to identify the first mammalian QTL for macronutrient-specific intake (carbohydrate and fat) and for total energy intake.  A region on proximal Chromosome 17 revealed two significant QTL that co-localized for increased macronutrient intake-carbohydrate (Mnic1) and total kilocalorie intake (Kcal2), adjusted for body weight.  An interval-specific congenic strain, B6.CAST-17, was then developed which verified the QTL.  A new sub-congenic strain was developed which retained the linked traits.  Important new findings emerged shows that this congenic interval confers an activity phenotype, i.e., mice carrying the differential segment have 20% higher spontaneous physical activity levels compared with the host B6 strain. We hypothesize that this Chromosome 17 QTL is either encoded by a single gene locus that determines both food intake and physical activity, or by two or more genes, each determining a sub-phenotype of energy balance.  Microarray analysis of skeletal muscle and hypothalamus in congenic and wild type B6 mice was carried out to identify potential candidate genes for the activity and food intake behavior.
Keywords: macronutrient-specific intake, sub-congenic strain

overall_design: Hypothalamus and skeletal muscle RNA were obtained from three male B6.CAST-17 homozygous congenic mice and three male wild type B6 mice. Each strain was represented by three mice, thus three biological replicates were analyzed for each strain.

first: Karunakaran

middle: G

last: Kumar

middle: K


title: Zhong You 821 Sclerotinia infected vs Mock infected controls in B. napus (Zhong You 821)

city: Saskatoon

first: Dwayne

last: Hegedus

state: SK

address: 107 Science Place

institute: Agriculture and Agri-Food Canada

summary:
The fungal pathogen Sclerotinia sclerotiorum infects a broad range of dicotyledonous plant species and is the causative agent of stem rot in Brassica napus. To elucidate the mechanisms underlying the defense response, we studied the patterns of gene expression in a partially resistant variety of ZhongYou 821 (ZY821) and a susceptible line from Westar over five time points, 6, 12, 24, 48, and 72 hours post-inoculation (hpi) using a B. napus oligonucleotide microarray. Maximum differential gene expression was observed at 48 hpi in both genotypes with ZY821 responding more quickly than Westar. Specific sets of genes exhibited higher levels of expression at the earlier stages of the infection (6-12 hpi), including genes encoding defense-associated proteins such as chitinases, glucanases, osmotins and lectins and genes encoding transcription factors belonging to the zinc finger, WRKY, AP2, and MYB classes. Genes encoding enzymes involved in JA, ethylene, and auxin synthesis were induced in both genotypes, as were those for gibberellin degradation. Changes in metabolic pathways affecting carbohydrate and energy metabolism appeared to be directed toward shuttling carbon reserves to the TCA cycle. Genes involved in glucosinolate and phenylpropanoid biosynthesis were highly up-regulated suggesting that secondary metabolites are also important components of the response to S. sclerotiorum in B. napus.

Keywords: Time course, infected vs mock infected

overall_design:
Time course experiment (6hr, 12 hr, 24 hr, 48 hr, 72 hrs), sclerotinia infected vs mock-infected. Biological replicates: 3, independently harvested.
Technical replicates: 2, dye swapped within each time for each biological replicate (total 6 slides per time point)
time after innoculation: 6 hours: zy 6h inf vs control rep1, zy 6h control vs inf rep1, zy 6h inf vs control rep2, zy 6h control vs inf rep2, zy 6h inf vs control rep3, zy 6h control vs inf rep3
time after innoculation: 12 hours: zy 12h inf vs control rep1, zy 12h control vs inf rep1, zy 12h inf vs control rep2, zy 12h control vs inf rep2, zy 12h inf vs control rep3, zy 12h control vs inf rep3
time after innoculation: 24 hours: zy 24h inf vs control rep1, zy 24h control vs inf rep1, zy 24h inf vs control rep2, zy 24h control vs inf rep2, zy 24h inf vs control rep3, zy 24h control vs inf rep3
time after innoculation: 48 hours: zy 48h inf vs control rep1, zy 48h control vs inf rep1, zy 48h inf vs control rep2, zy 48h control vs inf rep2, zy 48h inf vs control rep3, zy 48h control vs inf rep3
time after innoculation: 72 hours: zy 72h inf vs control rep1, zy 72h control vs inf rep1, zy 72h inf vs control rep2, zy 72h control vs inf rep2, zy 72h inf vs control rep3, zy 72h control vs inf rep3
biological replicate: zy 6h inf vs control rep1, zy 6h inf vs control rep2, zy 6h inf vs control rep3
biological replicate: zy 6h control vs inf rep1, zy 6h control vs inf rep2, zy 6h control vs inf rep3
technical replicate - labeled-extract: zy 6h inf vs control rep1, zy 6h control vs inf rep1
technical replicate - labeled-extract: zy 6h inf vs control rep2, zy 6h control vs inf rep2
technical replicate - labeled-extract: zy 6h inf vs control rep3, zy 6h control vs inf rep3
biological replicate: zy 12h inf vs control rep1, zy 12h inf vs control rep2, zy 12h inf vs control rep3
biological replicate: zy 12h control vs inf rep1, zy 12h control vs inf rep2, zy 12h control vs inf rep3
technical replicate - labeled-extract: zy 12h inf vs control rep1, zy 12h control vs inf rep1
technical replicate - labeled-extract: zy 12h inf vs control rep2, zy 12h control vs inf rep2
technical replicate - labeled-extract: zy 12h inf vs control rep3, zy 12h control vs inf rep3
biological replicate: zy 24h inf vs control rep1, zy 24h inf vs control rep2, zy 24h inf vs control rep3
biological replicate: zy 24h control vs inf rep1, zy 24h control vs inf rep2, zy 24h control vs inf rep3
technical replicate - labeled-extract: zy 24h inf vs control rep1, zy 24h control vs inf rep1
technical replicate - labeled-extract: zy 24h inf vs control rep2, zy 24h control vs inf rep2
technical replicate - labeled-extract: zy 24h inf vs control rep3, zy 24h control vs inf rep3
biological replicate: zy 48h inf vs control rep1, zy 48h inf vs control rep2, zy 48h inf vs control rep3
biological replicate: zy 48h control vs inf rep1, zy 48h control vs inf rep2, zy 48h control vs inf rep3
technical replicate - labeled-extract: zy 48h inf vs control rep1, zy 48h control vs inf rep1
technical replicate - labeled-extract: zy 48h inf vs control rep2, zy 48h control vs inf rep2
technical replicate - labeled-extract: zy 48h inf vs control rep3, zy 48h control vs inf rep3
biological replicate: zy 72h inf vs control rep1, zy 72h inf vs control rep2, zy 72h inf vs control rep3
biological replicate: zy 72h control vs inf rep1, zy 72h control vs inf rep2, zy 72h control vs inf rep3
technical replicate - labeled-extract: zy 72h inf vs control rep1, zy 72h control vs inf rep1
technical replicate - labeled-extract: zy 72h inf vs control rep2, zy 72h control vs inf rep2
technical replicate - labeled-extract: zy 72h inf vs control rep3, zy 72h control vs inf rep3

first: Jianwei

last: Zhao

first: Diana

last: Bekkaoui

first: Lone

last: Buchwaldt

first: Roger

last: Rimmer

first: Andrew

last: Sharpe

first: Linda

last: McGregor


title: Drosophila Gene Expression During Metamorphosis in Wild Type and Germline Minus Pupae

status: Public on Feb 11 2009

city: Los Angeles

first: Matthew

last: Lebo

address: 1040 Child's Way

department: Molecular and Copmutational Biology

institute: University of Southern California

summary:
	Drosophila melanogaster undergoes a complete metamorphosis, during which time the larval male and female forms transition into sexually dimorphic, reproductive adult forms. To understand this complex morphogenetic process at a molecular-genetic level, we performed whole genome microarray analyses. Genes were identified that were expressed during metamorphosis in both somatic and germline tissues of males and females.  Additionally, genes were identified that display sex-specific differences in abundance in both of these tissues at discrete times during metamorphosis.
Keywords: time course; wild type; genetic modification;

overall_design: Gene expression was examined at five time points during metamorphosis: 0, 24, 48, 71, and 96 hr After Puparium Formation (APF).  Gene expression was examined separately in males and females for both wild type pupae and tudor (tud) progeny.  tud progeny have genetically ablated germline tissues.  All samples were labeled with Cy5 and compared against a common reference sample labeled with Cy3.  The reference sample contained male and female wild type pupae from all stages of metamorphosis.  All experiments were conducted in triplicate.

first: Laura

last: Sanders

first: Fengzhu

last: Sun

first: Michelle

middle: N

last: Arbeitman


title: Sclerotinia infected vs Mock infected controls in B. napus (Westar)

summary:
The fungal pathogen Sclerotinia sclerotiorum infects a broad range of dicotyledonous plant species and is the causative agent of stem rot in Brassica napus.  To elucidate the mechanisms underlying the defense response, we studied the patterns of gene expression in a partially resistant variety of ZhongYou 821 (ZY821) and a susceptible line from Westar over five time points, 6, 12, 24, 48, and 72 hours post-inoculation (hpi) using a B. napus oligonucleotide microarray. Maximum differential gene expression was observed at 48 hpi in both genotypes with ZY821 responding more quickly than Westar.  Specific sets of genes exhibited higher levels of expression at the earlier stages of the infection (6-12 hpi), including genes encoding defense-associated proteins such as chitinases, glucanases, osmotins and lectins and genes encoding transcription factors belonging to the zinc finger, WRKY, AP2, and MYB classes. Genes encoding enzymes involved in JA, ethylene, and auxin synthesis were induced in both genotypes, as were those for gibberellin degradation.  Changes in metabolic pathways affecting carbohydrate and energy metabolism appeared to be directed toward shuttling carbon reserves to the TCA cycle.  Genes involved in glucosinolate and phenylpropanoid biosynthesis were highly up-regulated suggesting that secondary metabolites are also important components of the response to S. sclerotiorum in B. napus.

Keywords: Time course, and infected vs mock infected

overall_design:
Time course experiment (6hr, 12 hr, 24 hr, 48 hr, 72 hrs), sclerotinia infected vs mock-infected. Biological replicates: 3, independently harvested.
Technical replicates: 2, dye swapped within each time for each biological replicate (total 6 slides per time point)
time after innoculation: 6 hours: westar 6h inf vs control rep1, westar 6h control vs inf rep1, westar 6h inf vs control rep2, westar 6h control vs inf rep2, westar 6h inf vs control rep3, westar 6h control vs inf rep3
time after innoculation: 12 hours: westar 12h inf vs control rep1, westar 12h control vs inf rep1, westar 12h inf vs control rep2, westar 12h control vs inf rep2, westar 12h inf vs control rep3, westar 12h control vs inf rep3
time after innoculation: 24 hours: westar 24h inf vs control rep1, westar 24h control vs inf rep1, westar 24h inf vs control rep2, westar 24h control vs inf rep2, westar 24h inf vs control rep3, westar 24h control vs inf rep3
time after innoculation: 48 hours: westar 48h inf vs control rep1, westar 48h control vs inf rep1, westar 48h inf vs control rep2, westar 48h control vs inf rep2, westar 48h inf vs control rep3, westar 48h control vs inf rep3
time after innoculation: 72 hours: westar 72h inf vs control rep1, westar 72h control vs inf rep1, westar 72h inf vs control rep2, westar 72h control vs inf rep2, westar 72h inf vs control rep3, westar 72h control vs inf rep3
biological replicate: westar 6h inf vs control rep1, westar 6h inf vs control rep2, westar 6h inf vs control rep3
biological replicate:  westar 6h control vs inf rep1, westar 6h control vs inf rep2, westar 6h control vs inf rep3
technical replicate - labeled-extract: westar 6h inf vs control rep1, westar 6h control vs inf rep1
technical replicate - labeled-extract: westar 6h inf vs control rep2, westar 6h control vs inf rep2
technical replicate - labeled-extract: westar 6h inf vs control rep3, westar 6h control vs inf rep3
biological replicate: westar 12h inf vs control rep1, westar 12h inf vs control rep2, westar 12h inf vs control rep3
biological replicate:  westar 12h control vs inf rep1, westar 12h control vs inf rep2, westar 12h control vs inf rep3
technical replicate - labeled-extract: westar 12h inf vs control rep1, westar 12h control vs inf rep1
technical replicate - labeled-extract: westar 12h inf vs control rep2, westar 12h control vs inf rep2
technical replicate - labeled-extract: westar 12h inf vs control rep3, westar 12h control vs inf rep3
biological replicate: westar 24h inf vs control rep1, westar 24h inf vs control rep2, westar 24h inf vs control rep3
biological replicate:  westar 24h control vs inf rep1, westar 24h control vs inf rep2, westar 24h control vs inf rep3
technical replicate - labeled-extract: westar 24h inf vs control rep1, westar 24h control vs inf rep1
technical replicate - labeled-extract: westar 24h inf vs control rep2, westar 24h control vs inf rep2
technical replicate - labeled-extract: westar 24h inf vs control rep3, westar 24h control vs inf rep3
biological replicate: westar 48h inf vs control rep1, westar 48h inf vs control rep2, westar 48h inf vs control rep3
biological replicate:  westar 48h control vs inf rep1, westar 48h control vs inf rep2, westar 48h control vs inf rep3
technical replicate - labeled-extract: westar 48h inf vs control rep1, westar 48h control vs inf rep1
technical replicate - labeled-extract: westar 48h inf vs control rep2, westar 48h control vs inf rep2
technical replicate - labeled-extract: westar 48h inf vs control rep3, westar 48h control vs inf rep3
biological replicate: westar 72h inf vs control rep1, westar 72h inf vs control rep2, westar 72h inf vs control rep3
biological replicate:  westar 72h control vs inf rep1, westar 72h control vs inf rep2, westar 72h control vs inf rep3
technical replicate - labeled-extract: westar 72h inf vs control rep1, westar 72h control vs inf rep1
technical replicate - labeled-extract: westar 72h inf vs control rep2, westar 72h control vs inf rep2
technical replicate - labeled-extract: westar 72h inf vs control rep3, westar 72h control vs inf rep3


title: Gene expression of transgenic knock-in mutant growth hormone receptor mice

city: ST Lucia

first: Johanna

last: Barclay

state: Queensland

address: Building 80, University of Queensland

country: Australia

institute: Institute for Molecular Bioscience

summary:
A comparative study of gene expression in mice which carry different truncations or mutations of the growth hormone receptor
Keywords: genotype comparison

overall_design: Growth hormone receptor was truncated at amino acid 569, amino acid 391, mutated within the Box1 region, or removed altogether (KO) and transgenic mice were created.  At 4 months of age male mice were starved overnight and sacrificed.

first: Michael

last: Waters


title: cDNA aCGH study of pure DCIS without invasive tumor, DCIS associated with IDC and its IDC component

city: Toronto

first: Susan

last: Done

address: 610 University Ave

institute: Ontario Cancer Institute

summary:
cDNA aCGH study of pure DCIS (breast duct carcinoma in situ) without invasive tumor, DCIS associated with IDC (breast invasive duct carcinoma) and its IDC component
23 patients: 6 pure DCIS without invasive cancer and no history of invasive cancer, 17 DCIS associated with IDC. Out of the latter 1 tumor had only enough DCIS (#16) for aCGH and one - IDC (#23)
Keywords: Comparative clinical study

overall_design:
Histological sections microdissected, DNA extracted, subjected to whole genome amplification protocol (SCOMP) and aCGH.

The replicate raw data file GSM257548_2.gpr (dcis19_2.gpr) for GSM257548 is not available.

first: Vladimir

last: Iakovlev


title: Microarray-based comparative genomic profiling of reference strains of Actinobacillus pleuropneumoniae

city: Ottawa

first: John

last: Nash

state: Ontario

address: 100 Sussex Drive

department: Institute for Biological Sciences

institute: National Research Council

summary:
Microarray-based comparative genomic hybridizations were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines.

Keywords: Comparative Genomic Hybridisation

platform_organism: Actinobacillus pleuropneumoniae serovar 5b str. L20

overall_design: Microarray data for 15 known serotypes of Actinobacillus pleuropneumoniae (at least 2 replicates per serotype).

first: Wendy

middle: A

last: Findlay

first: Anne

last: Bouevitch


title: Comparative genomic profiling of reference strains and selected Canadian field isolates of A. pleuropneumoniae

status: Public on Jan 31 2009

city: St-Hyacinthe

first: Vincent

last: Deslandes

state: Quebec

address: 3200 rue Sicotte

department: Groupe de Recherche sur les Maladies Infectieuses du Porc

institute: Université de Montréal - Faculté de Médecine Vétérinaire

summary:
Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines.
Using hierarchical clustering, our M-CGH results showed that the majority of the genes in the genome of the serovar 5 A. pleuropneumoniae L20 strain were conserved in the reference strains of all 15 serovars and in representative field isolates. Fifty-eight conserved genes predicted to encode for outer membrane proteins or lipoproteins were identified. As well, there were several clusters of diverged or absent genes including those associated with capsule biosynthesis, toxin production as well as genes typically associated with mobile elements.

Keywords: Comparative Genomic Hybridization

overall_design: Two condition CGH experiments : tested strains vs reference 5bL20 strain.  26 strains were tested, with 2-4 technical replicates per strain, for a total of 74 samples.  One replicate per array.

first: Julien

last: Gouré

first: Simon

last: Foote

first: Janet

middle: I

last: MacInnes

first: James

last: Coulton

first: Mario

last: Jacques


title: Expression Profiling to Identify Novel Desiccation Response Transcripts from Tortula ruralis gametophytes

city: Lubbock

first: Paxton

last: Payton

state: TX

address: 3810 4th Street

institute: USDA-ARS

summary: In this study we have tried to utilize the unique aspects of the T. ruralis response to desiccation and rehydration to design a strategy to identify rehydrins that are of low abundance and perhaps completely novel to the desiccated or rehydration transcriptomes. We have constructed two Subtractive Suppression Hybridization (SSH) libraries (Diatchenko et al., 1996) that are designed to enrich for differentially expressed low-abundance transcripts contained within gametophytic cells either in the slow-dried state (mRNP sequestrated rehydrin transcripts) or cells that have been rapidly dried, rehydrated and sampled at 2h of hydration (rehydrin and recovery transcripts) when the translational change in gene expression is at its peak (Oliver 1991). To achieve this aim we constructed SSH libraries using PolyA RNA isolated from the polysomal (mRNP) fractions from the slow-dried and 2h rehydrated rapid dried gametophytes selected against PolyA RNA from hydrated control gametophytes as the source for driver cDNA. Collections of cDNA clones from each library were sequenced and used to generate a small T. ruralis SSH cDNA microarray for expression profiling of both total RNA extracts for transcript accumulation assessments and polysomal RNA extracts for transcript sequestration and recruitment assessments.

overall_design: To assess the expression characteristics of the transcripts represented by the SSH contigs we established a cDNA microarray containing the inserts (PCR derived fragment) from each of the 768 individual SSH ESTs, with the exception of thirteen that failed to generate a PCR fragment. Twelve of the missing thirteen SSH EST cDNAs were replaced with PCR fragments from previously isolated T. ruralis cDNAs four of which, representing the ribosomal proteins S14, S16, L23 and L15 (Wood et al., 2000, Zeng and Wood 2000), were previously reported to be constitutively expressed and were added to serve as normalization genes. The remaining eight clones, Tr155, Tr217, Tr403, Tr416, Tr421 (described by Scott and Oliver, 1994), and TrCDPK (U82087) were added as either positive “up-regulated” (Tr155, Tr403, Tr421), negative “down-regulated (Tr217, Tr416), or neutral (TrCDPK) controls based on previous northern analyses. The cDNAs were printed from two 384 well plates in 12 blocks (two columns of 6) of 24 x 8 spots such that each SSH EST and controls were represented in triplicate. Each of the triplicate cDNAs was separated within the blocks to eliminate possible spatial hybridization bias. All hybridizations were duplicated as dye swaps with two separate RNA preparations, from large populations of individual gametophytes (isolated from a minimum of three separate clumps), serving as the source for the sscDNA Cy3 and Cy5 labeled probes.  The RNA preparations for the Total polyA RNA were by necessity separate samples from those used to isolate Polysomal poly A RNA.

first: Melvin

last: Oliver

first: Jeremy

last: Hudgeons

first: Scot

last: Dowd


title: Effects of DEHP on the hepatic expression of a selection of mouse genes related to nuclear receptor signaling.

status: Public on Feb 19 2009

city: Villenave d'Ornon

first: Pascal

middle: GP

last: Martin

address: 71 avenue Edouard Bourlaux

department: UMR1332 BFP

institute: INRAE

summary:
Phthalates are industrial additives widely used as plasticizers. In addition to deleterious effects on male genital development, population studies have documented correlations between phthalates exposure and impacts on reproductive tract development and on the metabolic syndrome in male adults. In this study we investigated potential mechanisms underlying the impact of di-(2-ethylhexyl)-phthalate (DEHP) on adult mouse liver in vivo. A parallel analysis of hepatic transcript and metabolic profiles from adult mice exposed to varying DEHP doses was performed. Hepatic genes modulated by DEHP are predominantly PPARα targets. However, the induction of prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways, including Constitutive Androstane Receptor (CAR). Integration of transcriptomic and metabonomic profiles revealed a correlation between the impacts of DEHP on genes and metabolites related to heme synthesis and on the Rev-erbα pathway that senses endogenous heme level.

Keywords: Treatment effect

overall_design: One-color macroarrays, 4 experimental conditions: Control mice (vehicle treated), mice treated with di-(2-ethylhexyl)-phthalate (DEHP) at 30 mg/kg/day (D30), 180 mg/kg/day (D180) or 1100 mg/kg/day (D1100) for 14 days, Biological replicates: 6 controls, 4 D30, 4 D180, 5 D1100, One replicate per array

first: Alexandre

last: Eveillard

first: Frederic

last: Lasserre

first: Marie

last: De Tayrac


title: Two-component signal transduction systems in Streptomyces and related organisms

city: Taipei

first: Ralph

last: Kirby

address: Li-Nong Road

country: Taiwan

institute: National Yang-Ming University

summary: Two component sensor-response regulator systems (TCSs) are very common in the genomes of the Streptomyces species that have been fully sequenced to date. It has been suggested that this large number is an evolutionary response to the variable environment that Streptomyces encounter in soil. Notwithstanding this, TCSs are also more common in the sequenced genomes of other Actinomycetales when these are compared to the genomes of most other eubacteria. In this study, we have used DNA/DNA genome microarray analysis to compare fourteen Streptomyces species and one closely related genus to Streptomyces coelicolor in order to identify a core group of such systems. This core group is compared to the syntenous and non-syntenous TCSs present in the genome sequences of other Actinomycetales in order to separate the systems into those present in Actinomycetales in general, the Streptomyces specific systems and the species specific systems. Horizontal transfer does not seem to play a very important role in the evolution of the TCS complement analyzed in this study. However, cognate pairs do not necessarily seem to evolve at the same pace, which may indicate the evolutionary responses to environmental variation may be reflected differently in sequence changes within the two components of the TCSs. The overall analysis allowed subclassification of the orphan TCSs and the TCS cognate pairs and identification of possible targets for further study using gene knockouts, gene overexpression, reporter genes and yeast two hybrid analysis.

sample_organism:
Streptomyces antibioticus
Kitasatospora aureofaciens
Streptomyces bikiniensis
Streptomyces clavuligerus
Streptomyces exfoliatus
Streptomyces fradiae
Streptomyces griseus
Streptomyces microflavus
Streptomyces rochei
Streptomyces virginiae
Streptosporangium roseum
Streptantibioticus cattleyicolor
Streptomyces venezuelae
Streptomyces tanashiensis
Streptomyces coelicolor A3(2)
Streptomyces maritimus

overall_design: DNA/DNA comparative analysis using the University of Surrey PCR Microarray chip

first: Nai-hua

last: Hsiao


title: Drosophila Sex Hierarchy Regulated Gene Expression in 48 hour APF Pupae

first: Matthew

last: Lebo

summary:
Drosophila melanogaster undergoes a complete metamorphosis, during which time the larval male and female forms transition into sexually dimorphic, reproductive adult forms.  To understand this complex morphogenetic process at a molecular-genetic level, we performed whole genome microarray analyses.   In somatic tissues at 48 hour After Puparium Formation (APF), 173 sex-biased transcripts that likely function downstream of the doublesex (dsx) branch of the sex determination hierarchy were identified.  The mode of regulation of the sex-specific isoforms of DSX (DSX-F and DSX-M) was examined.  It was determined that for most downstream targets, DSX-F and DSX-M regulate gene expression in the same manner, but that one isoform acts as a more potent regulator.
Keywords: wild type; genetic modification

overall_design: All microarrays were dual channel with direct comparisons of male versus female or wild type versus mutant.  All samples consist of whole body pupae collected at 48 hour After Puparium Formation (APF).  For each experiment, four biological replicates were analyzed in a dye-swap design.

first: Laura

last: Sanders

first: Fengzhu

last: Sun

first: Michelle

last: Arbeitman


title: The molecular effects of cordycepin on RNA synthesis in Saccharomyces cerevisiae

status: Public on Jan 30 2009

city: Zürich

first: Bernahrd

last: Dichtl

address: Winterthurer Strasse 190

department: Institute of Molecular Biology

country: Switzerland

institute: University of Zürich

summary:
Cordycepin (3’ deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 3’ hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analysed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis. Unexpectedly, cordycepin treatment modulated 3’ end heterogeneity of ACT1 and ASC1 mRNAs and rapidly induced extended transcripts derived from CYH2 and NEL025c loci. Moreover, cordycepin ameliorated the growth defects of poly(A) polymerase mutants and the pap1-1 mutation neutralized the effects of the drug on gene expression. Our observations are consistent with an epistatic relationship between poly(A) polymerase function and cordycepin action and suggest that a major mode of cordycepin activity reduces 3’ end formation efficiency independently of its potential to terminate RNA chain elongation. Finally, chemical-genetic profiling revealed genome-wide pathways linked to cordycepin activity and identified novel genes involved in poly(A) homeostasis.

Keywords: response to drug treatment

overall_design: Each experiment was performed as triplicate. We analyzed RNA obtained from wild-type cells, from wild-type cells treated with 40 microgram/ml cordycepin for 1 hour, from pap1-1 mutant cells grown at permissive temperature (25°C) and from pap1-1 mutant cells grown at permissive temperature (25°C) treated with 40 microgram/ml cordycepin for 1 hour.

first: Sandra

last: Holbein

first: Bernhard


title: Gene expression based profiling of brains from mice infected with lethal Listeria monocytogenes over a 4-day period

city: Oklahoma City

first: Douglas

last: Drevets

state: OK

address: 921 NE 13th St

department: Medicine-Infectious Diseases

institute: University of Oklahoma Health Sciences Center

summary:
Data presented here show that gene expression in the mouse brain changes  during systemic infection relative to a steady state (uninfected control) mouse during systemic Listeria monocytogenes infection.  Microarray analysis of gene expression in the brain following i.v. inoculation of 1-2 LD 50 L. monocytogenes showed changes in brain transcriptional activity occurred prior to brain infection.
Keywords: infectious diseases model, gene expression profiling for cell signaling/trafficking

overall_design:
Mice were injected with 1-2 LD50 wild type L. monocytogenes or with sterile PBS (control) then seven animals from each group were harvested daily for 4 days.  From each day, groups of five infected animals were selected for microarray analysis based upon similarities in CFU bacteria in the spleen (day 1), blood (day 2), and brain (days 3 and 4) and were randomly paired with brains harvested from control animals.  RNA was extracted from the brain halves using the Atlas™ Pure Total RNA Labeling System and hybridized to plastic slides with 32P-labeled probes using Atlas™ Plastic Mouse 5K Microarray (Clontech, Mountain View, CA) according to the manufacturer’s directions.  Hybridization to the arrays and phosphorimaging analysis were performed in the OUHSC microarray facility.  Phosphorimaging analysis was performed using a Storm™ optical scanner (Molecular Dynamics Inc., Sunnyvale, CA), and initial background corrections were performed using the Array Vision Software (Imaging Research Inc., St. Catharines, Ontario).

Data analyses were performed using Genespring GX v. 7.3 (Agilent Technologies, Palo Alto, CA).  Raw signal data were normalized to the median of the entire chip for each sample, and all samples were further normalized per gene to the medians of data from the uninfected samples.  Data for each day were analyzed by Mann Whitney Wilcoxen nonparametric test with a p-value cutoff of 0.05.  Lists of genes were generated to reflect 2, 3, or 5-fold upregulation and downregulation based on significance.  Expression data were incorporated into Ingenuity software (Ingenuity Systems, Inc., Redwood City, CA) to enable further understanding of complex relationships as well as provide further information about key canonical pathways in which the genes of interest functions.

first: Jennifer

last: Schawang

first: Marilyn

last: Dillon

first: Megan

last: Lerner

first: Michael

last: Bronze

first: Daniel

last: Brackett


title: ChIP-chip analysis of NFI-1 in wildtype (N2) mixed stage C. elegans

city: Chapel Hill

first: Christina

middle: M

last: Whittle

address: 406 Fordham Hall, CB#3280

department: Biology

institute: University of North Carolina-Chapel Hill

type: Genome binding/occupancy profiling by genome tiling array

summary: The conserved Nuclear Factor I (NFI) family of transcription factors is unique to animals and essential for mammalian development. The C. elegans genome encodes a single NFI family member, whereas vertebrate genomes encode four distinct NFI protein subtypes (A, B, C, and X). NFI-1 deficient worms exhibit abnormalities including reduced lifespan, defects in movement and pharyngeal pumping, and delayed egg-laying. To explore the functional basis of these phenotypes, we sought to comprehensively identify NFI-1 binding targets in C. elegans. We first established NFI-1 DNA-binding specificity using an in vitro DNA-selection strategy. Analysis yielded a consensus motif of TTGGCA(N3)TGCCAA, which occurs 586 times in the genome, a 100-fold higher frequency than expected. We next asked which sites were occupied by NFI-1 in vivo by performing chromatin immunoprecipitation of NFI-1 followed by microarray hybridization (ChIP-chip). Only 55 genomic locations were identified, an unexpectedly small target set. In vivo NFI-1 binding sites tend to be upstream of genes involved in core cellular processes such as chromatin remodeling, mRNA splicing, and translation. Remarkably, 67/80 (84%) of the C. briggsae homologs of the identified targets contain conserved NFI binding sites in their promoters. These experiments provide a foundation for understanding how NFI-1 is recruited to unexpectedly few in vivo sites to perform its developmental functions, despite a vast over-representation of its binding site. To our knowledge, this represents the first genome-wide location study of any transcription factor in C. elegans, and the first genome-wide analysis in any species of an NFI transcription factor.

overall_design: 4 biological replicates of NFI-1 ChIP-chip (including 1 dye-swap) were performed with 2 pre-immune Mock ChIP-chip controls from corresponding extracts.

first: Elena

last: Lazakovitch

first: Richard

last: Gronostajski

first: Jason

middle: D

last: Lieb


title: The effects of Mycophenolate treatment on the transcriptomes of patients with symptomatic carotid artery stenosis

city: Amsterdam

first: Oscar

middle: Leonard

last: Volger

state: Noor Holland

address: Meibergdreef 15

department: Cardiology

institute: Academic Medical Center

summary: This study was conducted in order to assess the effects of the compound Mycophenolate mofetil (MMF), an inhibitor of the enzyme inosine monophosphate dehydroxygenase (IMPDH) with a strong cytostatic effect on T-cells, on the phenotype of human carotid artery stenosis in humans in vivo. This was done at the transcriptome level by comparing the gene expression profiles of carotid endarterectomy samples from patients with carotid artery stenosis (>70% diameter stenosis on angiography) that were randomly assigned to treatment with MMF 1000 mg (N=9) or Placebo (N=11), respectively. MMF or placebo was given for at least 2 weeks prior to undergoing carotid endarterectomy (CEA).

overall_design:
Patients and study personnel were blinded to study group assignments. The institutional ethics committee approved the study, and informed consent was obtained prior to enrollment. This study is registered as an International Standardised Randomised Controlled Trial, number ISRCTN84092396.
The transcriptomes were determined by means of hybridizing one round amplified and biotinylated cRNA molecules, extracted from the carotid endarterectomy samples, on Sentrix Illumina Human Ref-8 version2.0 gene expression chips.

first: Sander

last: van Leuven

first: Diederik

last: van Wijk

first: Jean-Paul

last: de Vries

first: Dominique

middle: V

last: de Kleijn

first: Chris

last: van der Loos

first: Anton

last: Horrevoets

first: Allard

last: van der Wal

first: Onno

last: de Boer

first: Gerard

last: Pasterkamp

middle: R

last: Hayden

first: John

last: Kastelein

first: Erik

last: Stroes


title: Ontoepigenetic profile of mouse liver reveals dynamic regulation of chromatin during development

status: Public on Jan 29 2009

city: Kansas City

first: Steven

last: Hart

state: KS

address: 3901 Rainbow Blvd

department: Pharmacology, Toxicology & Therapeutics

institute: University of Kansas Medical Center

type: Methylation profiling by genome tiling array

summary:
Fetal liver is predominately a hematopoietic organ, but shortly after birth transitions into the major site for xenobiotic (drug) metabolism.  The epigenetic mechanisms controlling this developmental switch remain an open door for exploration. DNA methylation (meDNA) governs many diverse cellular functions and is essential for normal development.  Histone H3 lysine 4 dimethylation (H3K4me2) is detected in active gene promoters and enhancers7.  Histone H3 lysine 27 trimethylation (H3K27me3) is involved in chromatin compaction around inactive genes and shows a dominant negative phenotype on gene expression.
Here, we used chromatin immunoprecipitation from mouse liver to determine the contributions of meDNA, H3K4me2, and H3K27me3 on facilitating a permissive chromatin state; allowing the transition from post-hematopoiesis to drug response.

BAR files that contain the intensities for all probes on the arrays. The intensities are expressed as signals estimating the fold enrichment relative to IgG input.  BED files contain custom tracks that can be directly uploaded to the UCSC Genome Browser (http://genome.ucsc.edu/) and contain 3 types of tracks: Intervals, Peaks, and Active Regions.  Interval Tracks show where the average signal intensity of all probes in an 800 bp sliding window are greater than the thresholds (3-fold enrichment for DNA methylation and 4-fold enrichment for histone modifications). Peak Tracks indicate the most enriched probe in a given interval.  The Active Regions Track shows genomic regions containing one or more overlapping intervals.

Keywords: Time Course

overall_design: By analyzing 14% of the mouse genome at four ages (embryonic day 17 [day -2], and postnatal days 1, 5, and 45), we found each modification occurs independently and can be considerably dynamic or age-specific after hematopoiesis but before adulthood.

first: S

first: X

last: Zhong


title: Genome-wide Analysis of Mono-, Di- and Trimethylation of Histone H3 Lysine 4 in Arabidopsis thaliana

first: matteo

last: Pellegrini

address: 621 Charles Young Drive South

department: MCD Biology

institute: UCLA

summary:
Background. Post-translational modifications of histones play important roles in regulating transcription by modulating the structural properties of the chromatin. In plants, methylation of histone H3 lysine4 (H3K4me) is associated with genes and required for normal plant development.

Results. We have characterized the genome-wide distribution patterns of mono-, di- and trimethylation of H3K4 (H3K4me1, H3K4me2 and H3K4me3, respectively) in Arabidopsis thaliana using chromatin immunoprecipitation and high-resolution whole-genome tiling microarrays (ChIP-chip). All three types of H3K4me are found to be almost exclusively genic, and two thirds of Arabidopsis genes contain at least one type of H3K4me in seedlings. H3K4me2 and H3K4me3 accumulate predominantly in promoters and 5’ genic regions, whereas H3K4me1 is distributed within transcribed regions. In addition, H3K4me3-containing genes are highly expressed with low levels of tissue specificity, but H3K4me1 or H3K4me2 may not be directly involved in transcriptional activation. Furthermore, a genome-wide preferential co-localization of H3K4me3 and H3K27me3 found in mammals does not appear to exist in plants, but H3K4me2 and H3K27me3 co-localize at a higher-than-expected frequency. Finally, the relationship between H3K4me and DNA methylation was explored by comparing the genome-wide distribution patterns of H3K4me1, H3K4me2 and H3K4me3 in wild type plants and the met1 DNA methyltransferase mutant.

Conclusions. H3K4me plays widespread roles in regulating gene expression in plants. Although many aspects of the mechanisms and functions of H3K4me appear to be conserved among all three kingdoms, we observed significant differences in the relationship between H3K4me and transcription or other epigenetic pathways in plants and mammals.

overall_design: Arabidopsis thaliana plants (accession Col) were grown on soil under continuous light for three weeks, and the aerial part of the seedlings were harvested. The met1-3 mutant plants were grown under the same conditions and harvested at a similar developmental stage. ChIP was performed as previously described using antibodies purchased from Abcam (anti-H3K4me1: ab8895; anti-H3K4me2: ab7766; anti-H3K4me3: ab8580; anti-H3: ab1791).  ChIP samples were amplified, labeled, and hybridized to microarrays as previously described (Zhang et al., 2007b; Zhang et al., 2006). Three biological replicates were performed for H3K4me1 and H3K4me3, and 6 biological replicates were performed for H3K4me2. For each H3K4me ChIP, an H3 ChIP was performed to isolate nucleosomal control DNA. Microarray hybridization intensities from probes that match a unique genomic region were analyzed using Tilemap with the Hidden Markov model option, as previously described (Ji and Wong, 2005)

first: Xiaoyu

last: Zhang

first: Yana

last: Bernatavichute

first: Shawn

last: Cokus

first: Matteo

middle: E

last: Jacobsen


title: Transcriptome of transition from primary to secondary stem development in Populus

city: corvallis

first: palitha

last: dharmawardhana

state: OR

address: 321 richarson hall,OSU

institute: OSU

summary:
The terminal region of the developing shoot provides a steep developmental gradient from primary to secondary growth that facilitates identification of the genes that play specialized functions during each of these phases of growth

Keywords: Developmental series

overall_design: Stem internodes excluding the nodes from apex to base of the tree were sampled for RNA extraction. Two individual trees were used as biological replicates

first: Palitha

last: Dharmawardhana

first: Steve

last: Strauss


title: Combined gene expression and network analyses and the role of the RNAP II C-Terminal Domain in mitotic segregation

city: Greenville

last: Stiller

address: East 5th Street

institute: East Carolina University

summary: The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA syntheses, coordinating an astounding array of protein-protein interactions. In yeast, mutations that separate established functional units of the CTD result in pleiotrophic effects, producing slow growth phenotypes and the accumulation of abnormally large cells.  In general, the downstream pathways for such CTD related phenotypes are uncharacterized and probably highly complex.  We also observed that a sizeable fraction of CTD mutants retain multiple buds on parent cells and are elongated relative to wild-type yeast.  FACScan analyses revealed a trend toward increased DNA content over multiple rounds of growth, indicating that insertions into the normal tandemly repeated CTD result in aneuploidy. To begin to address the role of the CTD in such complex changes in cell structure and behavior, we applied an integrated approach using the Saccharomyces cerevisiae Genome Database (SGD) and microarray data. We were able to identify candidate genetic networks that could explain chromosome missegregation and related phenotypic traits in CTD mutants. These networks provide links between CTD-associated proteins and kinetochore function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components.

overall_design:
The essential elements required for CTD function have been determined in yeast though substitution and insertion mutations (West and Corden 1995; Stiller and Cook 2004; Liu et al. 2008); the core CTD functional unit lies within tandem heptapeptides or “diheptads”.  In addition, CTD mutants with progressively longer polyalanine insertions between diheptads show a continuous decline in growth rates, and the induction of conditional phenotypes; this has been demonstrated out to five alanine (5A) insertions (Stiller and Cook, 2004).  Attempts to restore the global amino acid register via extending insertions to seven alanines between diheptad units proved to be lethal, leading to the conclusion that too great a separation between functional units puts undo stress on at least some essential CTD-protein interactions. (Liu et al, 2008).
During these prior investigations, we observed that mutants containing 5A insertions are, on average, twice as large as normal yeast, prompting us to investigate these cells in more detail.  Yeast cells were transformed with mutated CTD sequences containing 5 alanine insertions between diheptad units via the plasmid shuffle, as described in detail previously (West and Corden 1995; Stiller and Cook 2004).  For comparison yeast cells were transformed with YSPTSPS sequences via the plasmid shuffle, as described in detail previously (West and Corden 1995; Stiller and Cook 2004).
RNA was extracted from fresh pellets of yeast cultures at log phase, grown to an optical density of 0.8 at 600nm in 100 ml of YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30 ºC in a shaker at 225 rpm. Total RNA was extracted using Qiagen’s RNeasy Kit (Valencia, CA). Four replicates for each sample yeast strain (5A and wild-type CTD control) were prepared, and 10ug total RNA for every replicates were sent to Duke university DNA microarray center for further analyses. Array ID YO06N from Operon Yeast Genome Oligo Set version 1.1.2 was used for the hybridization. The direct labeling protocol was performed for sample RNA, which included steps of first strand synthesis, slide preparation, hydrolysis, cDNA purification, hybridization and array washing. Cy3 and Cy5 were used for labeling the samples. Maui hybridization and TIGR washing system were used in this protocol for array hybridization and washing respectively. The protocol can be found in Duke University microarray center website as http://www.genome.duke.edu/cores/microarray/services/spotted-arrays/protocols/.  Fluorescent DNA bound to the microarray was detected with a GenePix 4000B scanner (Axon Instruments, Foster city, CA), using the GenePix 4000 software package to locate signal from spots. Data processing was performed using Duke Univerisity BASE web server (https://base-server.duhs.duke.edu/). GO annotation was used for gene ontology.

first: Carlyle

last: Rogers

first: Zhenhua

last: Guo


title: The transcriptional pattern of Clostridium acetobutylicum following the downregulation of CAC0654 using antisense RNA

city: Newark

first: Eleftherios

middle: Terry

last: Papoutsakis

state: DE

address: 15 Innovation Way

department: Chemical Engineering

institute: University of Delaware

summary: Little is known regarding the entry into and the regulation of genes during the sporulation process of clostridia.  Using C. acetobutylicum as a model organism we designed an antisense RNA construct targeting the CAC0654 gene to investigate the role of this gene during sporulation and solvent formation.  Samples were taken throughout the growth phase and compared to a plasmid control strain to elucidate the transcriptional changes induced by the downregulation of the two-component system CAC0653 and CAC0654

overall_design: Samples were taken from a Bioflow II reactor at approximately 9, 12, 13.5, 15, 17, and 19 hrs after bioreactor inoculation containing strain 824(p654as). Two slides per timepoint were hybridized on a dye swap configuration. The control mRNA was generated by combining samples taken at 9, 12, 13.5, 15, 17, and 19 hrs hrs after bioreactor inoculation of the control plasmid strain, 824(pSOS95del). The replicate slide from the 12 hour sample showed high deviations and was discarded from the series.

first: Ryan

last: Sillers

first: Mohab

last: Al-Hanai

first: Carlos

last: Paredes

middle: T


title: miRNA profiles of tonsilar B and T lymphocytes

status: Public on Jan 28 2009

city: Pamplona

first: Raquel

last: Malumbres

state: Navarra

address: Avenida Pio XII 55

department: Hemato-Oncology

country: Spain

institute: University of Navarra/ Clínica Universidad de Navarra

summary:
miRNA profiling of human tonsilar B lymphocytes (naïve B cells, centroblasts and memory B cells) and tonsilar T cells, analyzing the 470 human miRNAs present in miRBase V9.1. B cell subpopulations and T cells were purified using specific antibodies, magnetic beads and the AutoMACS system (Miltenyi Biotec) from reactive tonsils obtained from routine tonsilectomies. Informed consents and the Institutional Review Board approval were obtained. Total RNA was extracted from cell pellets using the mirVana™ miRNA Isolation Kit (Ambion), and sent to LC Sciences facility for microarray hybridization using microfluidics technology. FirstChoice® Human Skeletal Muscle Total RNA (Ambion) was used as common reference for all hybridizations. Background substracted and normalized data from each channel was used to calculate log ratios sample/reference.

Keywords: miRNA profiling

overall_design: Dual channel experiments using FirstChoice® Human Skeletal Muscle Total RNA (Ambion) as common reference for all hybridizations. Reference was labeled with Cy5 and the lymphocyte extracted RNA with Cy3. Biological replicates: 4 for each B cell subpopulation (centroblasts, naïve and memory B cells) and 3 for T cells, all of them purified from reactive tonsils with specific antibodies and magnetic beads. Microarray hybridization was performed at LC Sciences, using slides containing in situ synthesized oligonucleotides to detect the 470 miRNAs present in miRBase V9.1. Probes hsa-miR-337 and hsa-miR-542-5p were excluded due to systematic dye bias, according to the manufacturer intructions. Background substracted and normalized detectable data was used to calculate log ratios sample/reference. Background is determined using a regression-based background mapping method.  The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots.  Raw data matrix is then subtracted by the background matrix. Normalization was carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data.   A transcript to be listed as detectable must meets at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5.  CV is calculated by (standard deviation)/(signal intensity).  When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level.

first: Izidore

middle: S

last: Lossos


title: Uterine stromal cell_cultured with uNK conditioned Medium

city: Cambridge

first: Andrew

last: Sharkey

address: Tennis Court Rd

department: Pathology

institute: University of Cambridge

summary:
The endometrium contains a distinct population of immune cells consisting of 70% natural killer (NK) cells that undergo cyclic changes during the menstrual cycle. However, how these uterine NK (uNK) cells interact with uterine stromal cells (SC) remains unclear. We therefore investigated the paracrine effect of medium conditioned by uNK cells on the gene expression profile of endometrial SC in-vitro using a cDNA Microarray. Our results, verified by real-time PCR and ELISA, reveal that soluble factors from uNK cells substantially alter endometrial SC gene expression. The largest group of up-regulated genes found were chemokines and cytokines, including IL-15 and IL-15Rα. The latter could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. In addition, the most abundantly up-regulated genes, including IL-8, CCL8 and CXCL1 have also been shown to be stimulated by contact of SC with trophoblast, suggesting that uNK cells work synergistically to support the initial trophoblast migration during implantation. Overall this study demonstrates for the first time the paracrine communication between uNK cells and uterine SC, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.
Keywords: Response of endometrial stromal cells to uNK conditioned medium

overall_design: This study was designed to identify the response of non-decidualised stromal cells from the endometrium, to soluble factors secreted by uterine NK cells.  Endometrial stromal cells were isolated from 7 patients and treated with control medium or medium conditioned by uterine Nk cells. THE 'REF' COLUMN ON EACH ARRAY IS THE SIGNAL PRODUCED BY A COMMON REFERNCE RNA SAMPLE THAT WAS LABELLED AS A SINGLE BATCH SAMPLE AND HYBRIDISED TO ALL THE ARRAYS- IT IS A 'COMMON REFERENCE'. THE 'TEST' SAMPLE COMPRISES EACH INDIVIDUAL SAMPLE OF CELLS TREATED AS DESCRIBED IN THE SERIES SUBMISSION. FOR EXAMPLE; SAMPLE Y1 (gsm2435820) IS RNA FROM PATIENT 1 TREATED WITH CONTROL MEDIUM, SAMPLE G1 (GSM245371) IS RNA FROM PATIENT 1 TREATED WITH NK CONDITIONED MEDIUM THESE TWO SAMPLES THEREFORE FORM A PAIR- IE CELLS FROM THE SAME PATIENT TREATED WITH CONTROL OR NK-CONDITIONED MIDIUM. Y2,G2 ARE FROM PATIENT 2, ETC

first: Ariane

last: Germeyer


title: Clostridium acetobutylicum ATCC 824 (pRDNA7) butyrate stress (0.6%)

summary: An intergenic region found to be enriched from a genomic library under butyrate stress was overexpressed and challenged with butyrate (0.6%). The overexpression strain was compared to the plasmid control to determine the transcriptional changes due to overexpression and butyrate stress.

overall_design: RNA samples were taken from both the overexpression strain (pRDNA7) and the plasmid control strain (pSOS95del) at 0, 15, 40, 120, 240, and 360 min post a 0.6% butyrate (pH 6.7) stress. Two slides per timepoint were hybridized on a dye swap configuration.

first: Jacob

last: Borden