title: Profile of Animal Cell Death Gene Expressing Nicotiana tabacum

last: Liu

summary:
Transgenic tobacco (Nicotiana tabacum) expressing Caenorhabditis elegans cell death genes, Ced4 and Ced3, show evidence suggesting such expressions protect the plants from infestation by the plant parasitic nematode Meloidogyne incognita.  Although positive results have been correlated with the gene expressions (data in preparation for publication; a draft of the publication can be provided upon request), the mechanism by which the nematode protection is manifested is not clearly understood.  One possibility is that the C. elegans cell death proteins produced by the transgenic plants are being ingested and incorporated into the nematode’s own cell death pathway, leading to their demise.  Alternatively, it is also possible that expression of the C. elegans cell death genes promotes the endogenous resistance genes of the plant, leading to nematode resistance.  We want to test the later hypothesis by conducting a reference design microarray experiment to establish the expression profile of Ced3, and Ced4 homozygous plants and Ced3xCed4 double heterozygous plants in comparison with wild-type tobacco plants.  If the hypothesis is correct, we expect to detect increased expression of pathogenicity-related genes in the transgenic plants.  Furthermore, characterization of the expression profiles in these transgenic plants will provide us directionality for our future research on the elucidation of this resistance mechanism.
Keywords: Reference design

overall_design: 27 hybs total

first: Calderon-Urrea

last: Alejandro


title: Application of Functional Genomics to the Study of Solanacea's Defense Mechanisms against Xanthomonas sp. Pathogenicity

status: Public on Sep 01 2007

city: Rockville

first: Jia

state: MD

address: 9712 Medical Center Drive

country: USA

institute: Plant Genomics

summary:
Xanthomonas campestris pv. campestris (Xcc) is a major bacterial pathogen of cruciferous plants worldwide. The pathogen produces polysaccharides including extracellular cyclic glucan, xanthan, and extracellular enzymes that are key virulence factors. Different Xcc mutants (8397-defective in xanthan and 8523- defective in extracellular glucans) have been obtained and characterized in previously, wich shown to be less infective than the wild type strain when inoculated in N.benthamiana, wich has shown to be an excellent model for the study of Xcc-plant interaction. The objective of this work is evaluate this compounds functions in the plant-pathogen interaction, in particular in the plant transcriptoma modulation to confer susceptibility or resistance to the infection.     Plant gene expression profiles would be obtained from independently inoculated leaves of N.benthamiana with the following strains of xanthomonas: Xcc. 8004 (wild type), Xcc. 8397 (xanthan minus), Xcc. 8523 (glucan minus), ant water (control). Leaves discs will be collected at 24 hs post infection and immediately submerged in liquid N2. Total RNA will be extracted with plant RNA specific kits (RNA easy QIAGen), treated with DNase, purified and quantified.
Keywords: Reference design

overall_design: 11 hybs total

first: Vojnov

last: Adrian


title: Molecular Markers for Predicting Potato Tuber Bruise Susceptibility

summary:
Blackspot bruising is the tuber discoloration resulting from mechanical damage in susceptible crops and is a major quality and environmental problem for the potato industry. The present study is aimed at identifying differences in gene expression which are correlated with the development of bruise susceptibility or resistance in tubers.  This may produce markers or diagnostic tests for use in predicting enhanced bruise susceptibility in field grown potato crops.  Different potato cultivars were grown in field plots at ADAS Gleadthorpe, Nottinghamshire, UK during three growing seasons – 2003, 2004 and 2005.  Random samples of tubers (8) were harvested by hand and cores excised from the subdermal tissues of the stolon end of the tubers and immediately frozen and stored in liquid nitrogen for RNA isolations.  The remainder of the crops were stored at 4oC for bruise susceptibility testing - samples of tubers (30) from each crop were impacted and incubated for 48h at 30oC.  Bruise susceptibility was scored, according to the intensity of pigmentation and the volume of tuber tissue affected, on a scale of 0 (very resistant) – 10 (very susceptible). Crops representing 'very susceptible', 'susceptible', 'moderate' and 'resistant' to bruising, were selected for RNA isolation.  Total RNA’s were isolated from frozen cores by the TIGR hot phenol extraction method, precipitated by LiCl and treated with DNase.  RNA concentrations were adjusted to ~2ug/ul and analysed on 1% TBE gels after formamide treatment. Any RNA’s which were not pure enough or showed degradation were discarded. Total RNA’s were pooled and 25ul samples aliquotted according to the required hybridisations indicated in the Excel spreadsheet and the on-line information form.
Keywords: Direct comparison

overall_design: 32 hybs total

first: Croy

last: Ron


title: Profiling gene expression levels to investigate the degree of substantial equivalence between GM and non-GM potato

summary:
The goal of this study was to calculate the degree of substantial equivalence between a GM potato line modified for late blight resistance and corresponding non-GM controls in the presence/absence of P. infestans inoculation. The GM phenotype was conferred through the introduction of the Rb gene which has been previously cloned from Solanum bulbocastanum (Song et al. 2003). The resulting GM phenotype was characterised by the expression of strong, partial resistance to P. infestans. By obtaining an expression profile from a GM and non-GM population before and after exposure to P. infestans, this effectively provides for the completion of two risk assessments: impact on the potato transcriptome of plus/minus genetic modification; before and after exposure to the pathogen.  For each biological replicate, individuals from three separate GM lines (A, B, C) and three separate non-GM lines (D, E, F) were randomly sorted and cultivated for analysis. Each GM line contained an independent, single transgene insertion, which was verified through Southern Blot analysis. One day prior to pathogen exposure, the 4-week-old potato plants were transferred into a growth chamber with a 16-h light period, 90% relative humidity at a temperature of 15C. Plants were randomly selected and 14 individuals/line were inoculated with a field isolate of P. infestans. An additional 14 plants/line were sprayed with water. Inoculations were made with a sporangial suspension of 20,000/mL and plants were sprayed until run-off. Twenty-four hours post-inoculation, leaf tissue was collected from 9 plants per treatment, flash frozen in liquid nitrogen and pooled. Total leaf RNA was extracted using Trizol and the whole experiment was repeated three times (three biological replicates).
Keywords: Direct comparison

overall_design: 19 hybs total

first: Mullins

last: Ewen


title: Non-additive and imprinted gene expression in hybrid maize endosperm_13DAP

city: Saint Paul

first: Nathan

middle: M

last: Springer

state: MN

address: 1445 Gortner Ave

department: Plant Biology

institute: University of Minnesota

summary:
The nuclear content of the plant endosperm is the result of the contribution two maternal genomes and a single paternal genome.  This 2:1 dosage relationship provides a unique system for studying the additivity of gene expression levels in reciprocal hybrids.  A combination of microarray profiling and allele-specific expression analysis was performed using RNA isolated from endosperm tissues of maize inbred lines B73 and Mo17 and their reciprocal hybrids at two developmental stages, 13 and 19 days after pollination.  By assessing the relative levels of expression in the reciprocal hybrids it was possible to determine the prevalence of additive and non-additive expression patterns.  While the majority of differentially expressed genes displayed additive expression patterns in the endosperm, approximately 10% of the genes displayed non-additive expression patterns including maternal-like, paternal-like, dominant high-parent, dominant low-parent and expression patterns outside the range of the inbreds.  The frequency of hybrid expression patterns outside of the parental range in maize endosperm tissue is much higher than that observed for vegetative tissues.  For a set of 90 genes allele-specific expression assays were employed to monitor allelic bias and regulatory variation.  Eight of these genes exhibited evidence for maternally or paternally biased expression at multiple stages of endosperm development and are potential examples of differential imprinting.  Collectively, our data indicate that parental effects on gene expression are much stronger in endosperm than in vegetative tissues, and that endosperm imprinting may be far more common than previously estimated.
Keywords: genotype comparison

overall_design: Affymetrix microarrays were used to perform expression profiling on 13 day after pollination endosperm tissue of four different genotypes; B73; Mo17, B73xMo17 and Mo17xB73.  There are three biological replicates for each of the tissues.  Each biological sample represents a pool containing 5 endosperms each, from 6 different ears.

first: Robert

last: Stupar

first: Peter

middle: J

last: Hermanson


title: Host responses of Nicotiana benthamiana to infection by multiple plant viruses

summary:
Objectives: Our work focuses on the responses of Solanaceous plants to viruses that cause economically important diseases in tree fruits.  Using mock inoculated leaf tissue as a reference, we plan to compare the gene expression profiles of Nicotiana Benthamiana plants infected with one of three viruses; Plum Pox Potyvirus (PPV), Tomato Ringspot Nepovirus (ToRSV), and Prunus Nectrotic Ringspot Nepovirus (PNRSV).  Our goals are as follows: (1) Identify genes that are induced/repressed in response to individual viruses. (2) Identify genes that are induced/repressed in response to all 3 viruses. (3) Compare results to existing potato array data to look for similarities in responses to other pathogens.  Experimental Design: Nicotiana benthamiana plants were inoculated with one of three viruses: PPV, ToRSV, or PNRSV.  3 week old plants were inoculated by rubbing virus infected plant sap onto leaves dusted with carborundum.  Control plants were mock inoculated using sap from healthy plants.  All plants were maintained in a growth chamber at 22C for 18 days.  8 plants were inoculated with each virus or mock inoculated.  This experiment was repeated twice.  4 biological replicates derived from 2 virus infected plants from each replica experiment (4 plants) are to be used for hybridizations.  RNA from all mock inoculated plants was similarly pooled to create 4 biological replicates.  Each replicate control will serve as a universal reference sample that is to be hybridized pair wise with each of the three virus infected samples.  RNA extraction: After 18 days, un-inoculated leaves displaying clear symptoms were harvested and immediately frozen in liquid N2.  Total RNA was purified using Trizol according to TIGRs listed protocol.  RNA was subsequently treated with Turbo DNA-free RNase (Ambion cat#1907).  Finally, total RNA was further purified on RNeasy columns (Qiagen) according to manufacturer’s instructions and quantified using a Nanodrop spectrophotometer.
Keywords: Reference design

overall_design: 23 hybs total

first: Dardick

last: Christopher


title: Non-additive and imprinted gene expression in hybrid maize endosperm_19DAP

overall_design: Gene expression levels were profiled in 19 day after pollination endosperm tissue from four maize genotypes; B73, Mo17, Mo17xB73 and B73xMo17.


title: Quantitative resistance response to late blight in potato

summary:
Whole plants of a clone of S. cajamarquence and a clone of S. tuberosum were spray inoculated with Phytophthora infestans sporangial suspension 3000 sporangias/ml until run off. Two isolates were used: PE84006 (race 0) and POX067 (avr 8, 9). Leaves in the middle part of the plants were collected at 72 h and 96 h after inoculation. Leaves of untreated plants were collected as control treatment.
Keywords: Direct comparison

overall_design: 24 hybs total

first: Lindqvist-Kreuze

last: Hannele


title: Global analysis of gene expression associated with germination in tomato seeds.

summary:
Treatment of the seeds. The Solanum lycopersicum var. Money Maker wild type (MM) and fri1-1 mutant seeds were incubated on distilled-water saturated cotton wool in clear plastic boxes wrapped in black plastic sheets in darkness during 3 hours at 25°C. After that, the seeds were treated as follows: a) 24 hr of continuous irradiation with far-red light, FRc; b) 24 hr of FRc follow by a 10 min red light pulse (Rp), FRc+Rp; and c) complete darkness. After the light treatments, the seeds were incubated in darkness (25°C) until sampling.   Red light (35umol/m2sec) was provided by 40W fluorescent lamps (Phillips 40/15) in combination with a red translucid acrylic. FRc light (100 umol/m2sec) was provided by a set of 150W incandescent internal reflector lamps (Phillips) in combination with a red acetate filter, six 2mm thick blue acrylic filters Paolini 2031 and 10cm water filter.  Total RNA isolation. The seeds were grinded in liquid nitrogen and extracted with two volumes of extraction buffer (1% SDS, 6% p-aminosalycilic acid, 1% NaCl, 6% water-saturated phenol and 2% 2-mercaptoethanol) and two volumes of acid phenol:chloroform solution. The aqueous phase was sequentially extracted with one volume of acid phenol:chloroform solution and one volume of chloroform. The nucleic acids in the aqueous phase were precipitated with 0.15 volumes of 3M NaCl and 2.5 volumes of 100% ethanol at –80°C for 20 minutes. After centrifugation, the pellet was washed with 70% ethanol, resuspended in RNase-free water and precipitated with one volume of 4M LiCl over night at 4°C. The LiCl was removed after centrifugation and the pellet was rinsed with 2M LiCl and resuspended in RNase-free water. The total RNA was treated with RNase-free DNase (Promega) and cleaned up with mini spin columns (RNeasy Plant Mini Kit, Qiagen). The mRNA was amplificated with Message Amp II aRNA Amplification Kit (Ambion) and concentrated by vacumm centrifugation.
Keywords: Direct comparison

overall_design: 21 hybs total

first: Botto

last: Javier


title: Solanaceae comparative gene expression profiling

summary:
Six different Solanaceae species, Potato (Solanum tuberosum), Tomato (Lycopersicum esculentum), Pepper (Capsicum annuum), Tobacco (Nicotiana tabacum), Petunia and Nicotiana benthamiana were grown at 25C, 16h light and 8h darkness. Mature leaves were harvested after 4-6 weeks. RNA was isolated using Qiagen RNeasy. Tomato, pepper, petunia tobacco and N. benthamiana samples were hybridized against potato samples.
Keywords: Direct comaprison

sample_organism:
Capsicum annuum
Solanum lycopersicum
Nicotiana tabacum
Nicotiana benthamiana
Petunia x hybrida
Solanum tuberosum

overall_design: 10 hybs total

first: Rensink

last: Willem


title: Drought Stress Response of Andean Potato

summary:
The response to moderate and heavy drought of two Solanum tuberosum ssp. Andigena varieties, Sullu (NP 03.03) and SA 2563 (NP 03.01), planted in rain- and soil water protected fields in the Peruvian highlands are compared. Previous experiments indicate that Sullu has a greater capacity for yield maintenance under drought than SA 2563. Both clones have similar morphological properties, vegetative periods and rooting depths, so it can be assumed that the cause for increased drought tolerance of clone NP 03.03 is rather due to physiological or biochemical mechanisms, than to drought escape by deep rooting or earliness. Sullu and SA 2563 were planted in a random block design with 5 plants per bloc and 7 repetitions per treatment.  Treatments: (1) drought stress, (2) irrigated control The plants were drip-irrigated in both treatments until tuberization (until day 84 after planting). Subsequently, the irrigation was stopped in the drought field, but continued in the control field. The soil moisture content in the control field was kept near field capacity.   Planting date: October 05 2004 Start of drought treatment (during tuberization, 84 days after planting): December 28 2004  First sampling (soil water potential: -0.3 mPa 114 days after planting): January 27 2005 Second sampling (soil water potential –0.6 MPa, 134 days after planting): February 15 2005 Harvest: March 19 2005 (165 days after planting)  The experimental design includes gene expression analysis in leaves, roots and stolons at two time points, when soil water potential reaches -0.3 and –0.6 MPa. Gene expression changes will be set in relation with physiological and agronomical data obtained in the same experiment.
Keywords: Direct comparison

first: Schafleitner

last: Roland


title: Exploring Biodiversity in Andean Potato Varieties

summary:
Seed of 4 lines of S. tuberosum var andigena were sown and, after transplanting, grown in 3 gal nursery containers in a greenhouse with natural daylight.  The seeds were sown in July and the drought stress experiment began in September.  Drought stress was administered by withholding water and monitored by measuring the rate of photosynthesis (PS; LiCor 6400).  We found that loss of photosynthetic capability (ie a PS rate of 0-2 mM CO2/m2/sec) correlated with a severe drought stress.  Control plants were watered normally and maintained a PS rate of 18-20 mM CO2/m2/sec.  After drought stress, the treated plants were re-watered and PS measurements taken again.  After the first cycles of stress, control and treated plants were harvested and roots, tubers and shoots were stored at –80°C for RNA extractions.  The drought experiment was then repeated for the remaining plants such that they were exposed to a second cycle of stress.  For each line of S. andigena, there were 2 control and 2 treated plants per cycle of stress.  RNA was extracted following the acid phenol protocol of TIGR.
Keywords: Direct comparison, loop design

overall_design: 30 hybs total

first: Grene

last: Ruth


title: Tomato response to TMV after 1h of infection

summary:
TMV-resistant, TMV-susceptible and enhanced susceptibility mutant tomato were grown in asceptic conditions.  At approximately 5 weeks, leaves were mock inoculated, TMV inoculated, or left untreated.  Leaf tissue was collected 1hour after treatment.  RNA was isolated and purified; hybridizations were performed using 130ug of total RNA from plants mock inoculated versus TMV inoculated and untreated.  Each RNA sample is from two pooled plants.  Two replicates were completed.
Keywords: Direct comparison

overall_design: 12 hybs total

first: Yamamoto

last: Miki


title: Potato responses to cold, heat and salt stress

summary:
Potato seedlings were subjected to cold, heat and salt stress. Expression profiles were captured at three different time-points, 3h, 9h and 27h from two different tissues, roots and leaves. The experiment was preformed independently three times. Commercially available true potato seeds (Variety Gilroy) were germinated on rafts floating on hydroponic medium in Magenta boxes. Plants were grown for 5 weeks prior to stress application under long day conditions (16h light and 8h dark) at 25C with gentle agitation. To initiate stress the medium was replaced with fresh medium pre-chilled to 4C (cold stress), pre-heated to 35C (heat stress) or supplemented with 100mM NaCl (salt stress). Cold and heat stress were maintained for the duration of the experiment by placing the Magenta boxes on ice or in a water-bath at 35C. For every individual sample two boxes of plants were used pooling a total of 6 plants per sample. For each time-point a single control sample was used by changing the media in a similar way as for the stress induction. A total of six boxes were combined for the pooled reference samples. Plants were harvested at the appropriate time and snap-frozen in liquid nitrogen. Roots and aerial tissue was separated prior to freezing. The tissue was stored at -80C freezer until isolation. Total RNA was isolated using RNeasy isolation kit. The intactness of the RNA was verified on gel and the concentration was adjusted to 3ug/ul by ethanol precipitation and re-suspension.
Keywords: Direct comparison

overall_design: 72 hybs total


title: Tomato responses to the whitefly-transmitted TYLCV

summary:
Female whiteflies are used to inoculate the susceptible and resistant tomato genotypes as follows. Whiteflies are caged with TYLCV-infected tomato plants for a 48 h acquisition access period. Then, the viruliferous insects are collected and used to inoculate 20 plants of each genotype. The plants at their 4 leaf-stage are inoculated by caging ten viruliferous whiteflies with the youngest true leaf (more than 1 cm) of each plant, using leaf cages. Mock inoculations are conducted using the same number of non-viruliferous insects. After a 12 h inoculation access period (which ensures 100% infection), the insect cages are removed and the plants are treated with insecticide (imidacloprid). The plants are kept for 3 or 7 days in an insect-proof greenhouse, in natural daylight (12 h light/12 h dark).
Keywords: Direct comparison, loop design

first: Czosnek

last: Henryk


title: Mi-1-mediated defense response to nematodes and aphid

summary:
A pair of nearly isogenic tomato (Solanum lycopersicum) lines were used. These are: Motelle (Mi/Mi), root-knot nematode resistant and Moneymaker (mi/mi) root-knot nematode susceptible. A potato aphid (red biotype), imported from France, was used for infestation. An equal volume of sterile 1% carboxymethyl cellulose in water was added to the cleaned nematodes to keep them in suspension. The inoculum, consisting of 75-100 J2s, was dispensed in 10 µl aliquots using a repeat pipetter.  The inoculum was pipetted 2 mm beneath the root tips. Control seedlings were inoculated with 0.5% carboxymethyl cellulose.  After the desired incubation time (24 h or 36 h), seedlings were taken out of the boxes and a few set aside to check nematode infection rate. In our hand, this system results in 50% infection rate. The rest of the seedlings were placed on a water-saturated filter paper and the roots aligned.  About 1 cm of the infected root tip was excised with a blade and quickly frozen on a polycarbonate membrane-lined 50 mm diameter Petri dish floating on liquid nitrogen and stored at -80C. Plants were grown for 6 weeks in a growth chamber under 16 h light/8 h dark at 25C in UCMix/sand, irrigated with water or MiracleGroR solution, then moved to an insect growth room (25C) 4-6 days before aphid infestation. Individual tomato leaflets were infested with 30-40 mixed stages of the potato aphids using plastic leaf cages. Four leaflets and two plants of each genotype were infested. Two plants of each genotype were used as controls. After the desired infestation time (6, 12, 24 or 48 h), leaflets were excised from the plants with a blade and sprayed with 1% SDS to force aphids to remove their stylets from the leaf tissue.  Aphids were carefully removed from the leaflets and quickly frozen in liquid nitrogen. Control leaflets were excised and sprayed with 1% SDS before freezing. Samples were stored at -80C. RNA was isolated using the hot phenol method.
Keywords: Direct comparison

overall_design: 28 hybs total

first: Kaloshian

last: Isgouhi


title: Response of tomato leaves to tobacco mosaic virus (TMV)

last: Liu

summary:
TMV-resistant (N), -susceptible (n), and enhanced susceptible mutant sun1-1 (N) tomato were grown in asceptic conditions in growth chambers (25º, 16hr/8hr light dark).  4-5 week old tomato were treated with TMV leaf sap or mock leaf sap, and leaf tissue was collected after 3, 9, and 27 hours.    RNA was isolated from each sample (leaf tissue from one plant/one treatment/one timepoint), using standard TIGR protocols, and 25ug of total RNA was labeled using TIGR indirect labeling protocols.  The experiment was repeated three times.
Keywords: Direct comparison


title: Non-coding antisense transcription detected by conventional and single-stranded cDNA microarray

city: Lund

first: Johan

last: Staaf

address: Medicon Village

country: Sweden

institute: SCIBLU - Swegene Centre for Integrative Biology at Lund University

summary:
Recent bioinformatic studies revealed that many mammalian protein-coding genes also transcribe their complementary strands. This phenomenon raises questions regarding the validity of data obtained from double-stranded cDNA microarrays since hybridization to both strands may occur. To analyze experimentally the incidence of antisense transcription in human cells and to estimate their influence on protein coding expression patterns obtained by double-stranded microarrays, we profiled transcription with sense and antisense independently by using strand-specific cDNA microarrays.
Keywords: Common reference design

overall_design: We modeled an experimental design typically used for tumor classification studies. We performed hybridizations using direct-labeled randomly primed total RNA extracted from eight breast-cancer cell lines and one derived from normal breast epithelium against Universal Human Reference RNA as a common reference following standard protocols.

last: Vallon-Christersson

first: Anders

last: Kvist

first: Patrik

last: Medstrand

first: Ake

last: Borg

first: Carlos

last: Rovira


title: A transcript profile of Medicago truncatula 2HA and Jemmalong during somatic embryogenesis

city: Canberra

first: Peta

last: Holmes

state: Australian Capital Territory

address: Building 46, Australian National University

department: Reseach School of Biological Sciences

country: Australia

institute: Genomic Interactions Group

summary:
The Medicago truncatula line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than wild-type Jemalong. We have compared transcriptomes of tissue cultures from leaf explants of these two lines.
We have used the Affymetrix Medicago Genome Array GeneChip to compare leaf explants of Medicago truncatula Jemalong and the superembryogenic line 2HA after two weeks of tissue culture on 10 µM 1-naphthaleneacetic acid (NAA) and 4 µM 6-benzylaminopurine (BAP) (P4 10:4)
Keywords: Genotype comparison

platform_organism:
Sinorhizobium meliloti
Medicago sativa
Medicago truncatula

overall_design: Leaf explants from the two genotypes were treated with 10:4 for two weeks; total RNA was extracted and used for hybridization to Affymetrix arrays

first: Nijat

last: Imin

first: Nicolas

last: Goffard


title: Deletion mapping by the AtMap1 array

city: Kitashirakawa, Sakyoku

first: Ikuko

last: Hara-Nishimura

state: Kyoto

address: Oiwakecho

department: Depertment of Botany

country: Japan

institute: Kyoto University

type: Genome variation profiling by genome tiling array

summary:
We designed a novel tiling array, AtMap1, for genomic deletion mapping. The AtMap1 is a 60-mer oligonucleotide microarray consisting of 42,497 data probes designed from the genomic sequence of Arabidopsis thaliana Col-0. The data probes are almost evenly distributed over the genome except around centromeres. The average probe interval is 2.8 kb. The performance of the AtMap1 array was assessed using the mag2-2 mutant, which is a well-studied deletion mutant. Eight of the probes showed 3-fold lower signals in mag2-2 than Col-0. Seven of these probes were located in one region on chromosome 3. We treated these adjacent probes as one deletion. This deletion included previously reported deleted region. The other probe was located near the end of chromosome 4. A deletion around the probe was confirmed by PCR. This deletion was newly identified in this study. Thus, we concluded that the AtMap1 array was sufficiently sensitive to identify a deletion without a priori knowledge. From an analysis using the result of hybridization of Ler and previously reported polymorphism data, we found a tendency that the signal decrease depended on the overlap size of sequence polymorphisms. Mutation mapping is time-consuming, laborious and costly. The AtMap1 array removes the difficulties. The AtMap1 array is useful especially for mutations for which linkage-based mapping is difficult, e.g., mutations whose phenotype is highly variable among ecotypes, mutations whose phenotype is difficult to measure and revertant mutations.
Keywords: comparative genomic hybridization

overall_design:
This series includes:
Three self-comparison experiments to assess the reproducablity of the AtMap1 array.
Comparison of Col-0 and mag2-2, which is a well-studied deletion mutant induced by T-DNA insertion.
Comparison of Col-0 and rot3-1, which is a well-studied deletion mutant induced by fast-neutron irradiation.
Comparison of Col-0 and zig-2, which is a well-studied deletion mutant induced by fast-neutron irradiation.
Comparison of Col-0 and Ler, which is one of A. thaliana ecotypes.
Comparison of different sets of restriction enzymes for genome digestion.

first: Atsushi

last: Nagano


title: Listeria monocytogenes Cold Regulon in Log- and Stationary-Phase cells

status: Public on Aug 31 2007

city: Ithaca

first: Sarita

last: Raengpradub

state: NY

address: 412 Stocking Hall

department: Food Science

institute: Cornell University

summary:
	As the foodborne pathogen Listeria monocytogenes has the ability to grow at refrigeration temperatures, whole-genome microarray experiments were performed using L. monocytogenes strain 10403S to define the cold stress regulon and to identify genes differentially expressed during growth at 4°C and 37°C.  Microarray analysis using a stringent cutoff (adjusted p<0.001; fold-change >2.0) revealed 105 and 170 genes that showed higher transcript levels in logarithmic- and stationary-phase cells, respectively, at 4°C (compared to cells at 37°C).  A total of 74 and 102 genes showed lower transcript levels in logarithmic- and stationary-phase cells grown at 4°C, respectively.  Genes upregulated at 4°C during both stationary- and log-phase included those encoding a two-component response regulator (lmo0287), a cold shock protein (cspL), and two RNA helicases (lmo0866 and lmo1722), whereas genes encoding selected virulence factors and heat shock proteins were downregulated at 4°C.  Selected genes that were upregulated at 4°C during both stationary- and log-phase were confirmed by quantitative reverse transcriptase PCR.  Our data show (i) a large number of L. monocytogenes  genes are differentially expressed at 4 and 37°C with a larger number of genes showing higher transcript level at 4°C than genes showing lower transcript levels at 4°C; (ii) L. monocytogenes genes upregulated at 4°C include a number of genes and operons with previously reported or plausible roles in cold adaptation; and (iii) L. monocytogenes genes downregulated at 4°C include a number of virulence and virulence-associated genes as well as some heat shock genes.
Keywords: Listeria monocytogenes, cold regulon, temperature

platform_organism:
Listeria monocytogenes
Listeria innocua

overall_design: Independent RNA isolations were performed for each growth experiment (37°C  and 4°C , log and stationary phase). Three biological replicates were used in competitive whole-genome microarray experiments. For each set of hybridizations (log and stationary phase), RNA from cells cultured at 37°C were hybridized to RNA from cells cultured at 4°C.

first: Yvonne

middle: C

last: Chan

first: Kathryn

last: Boor

first: Martin

last: Wiedmann


title: Global Transcriptional Responses to Cisplatin in Dictyostelium discoideum Identify Potential Drug Targets

city: Houston

first: Nancy

middle: E

last: Van Driessche

state: TX

address: One Baylor Plaza

institute: Baylor college of medicine

summary:
Dictyostelium discoideum is a useful model for studying mechanisms of cisplatin drug sensitivity. Our previous findings, that mutations in sphingolipid-metabolism genes confer cisplatin resistance in D. discoideum and in human cells, raised interest in the resistance mechanisms and their implications for cisplatin chemotherapy. Here we used expression microarrays to monitor physiological changes and to identify pathways that are affected by cisplatin treatment of D. discoideum. We found over 400 genes whose regulation was altered by cisplatin treatment of wild type cells, including groups of genes that participate in cell proliferation and in nucleotide and protein metabolism. These findings show that the cisplatin response is orderly and multifaceted. Transcriptional profiling of two isogenic cisplatin-resistant mutants, impaired in different sphingolipid metabolism steps, showed that the effect of cisplatin treatment was greater than the effect of the mutations, indicating that cisplatin-resistance in the mutants is due to specific abilities to overcome the drug effects rather than to general drug insensitivity. Nevertheless, the mutants exhibited significantly different responses to cisplatin compared to the parent and over 200 genes accounted for that difference. We mutated some of the cisplatin-response genes and found that the mutants had altered drug sensitivity. These findings reveal the power of this system to identify pathways and genes that are affected by drugs and mutations. Our data illustrate how modeling complex cellular responses to drugs in genetically stable and tractable systems can uncover new targets with the potential for improving chemotherapy.
Keywords: Dictyostelium discoideum, cisplatin treatment, transcriptional profiles, physiological changes

overall_design: Exponentially growing cells (2x10E6 cells/ml) were divided into twelve 50 ml aliquots in 500 ml flasks. The cells were shaken for 30 min, 300 µM cisplatin was added to 6 cultures (PT buffer was added to the 6 controls), and the cells were shaken for 3 hr at 22C in the dark. The cells were washed with phosphate buffered saline (PBS) and resuspended in 1.0 ml TRIzol reagent (Life Technologies, Gaithersburg, MD) and RNA was extracted. For technical variation, RNA samples from each experiment were hybridized to at least 5 arrays (technical replication). For biological variation, each treatment (with or without cisplatin) was repeated twice for each mutant and 6 times for the wild type (biological replication). We analyzed both wild type and 2 mutant strains that have a higher resistance to cisplatin than wild type.

first: Hannah

last: Alexander

first: Junxia

last: Min

first: Adam

last: Kuspa

first: Stephen

first: Gad

last: Shaulsky


title: pH stress in Desulfovibrio vulgaris Hildenborough

city: Berkeley

middle: P.

last: Arkin

state: CA

address: 1 Cyclotron Road

department: Physical Biosciences Division

institute: Virtual Institute for Microbial Stress and Survival

summary:
Using data from microarray experiments, we investigated the effects of excess NaCl on D. vulgaris.
Keywords: stress response, time course

sample_organism: Nitratidesulfovibrio vulgaris str. Hildenborough

overall_design: Comparison of cells treated with pH10 pH to untreated cells at times of 0,120,240,30,60 min.


title: The effect of RPN9 silencing in N. benthamiana

first: Jia

last: Liu

summary:
N. benthamiana plants were grown under 16 hour light/8 hour dark cycle in a plant growth room at 24°C for approximately six weeks before subjected to virus-induced gene silencing. Agrobacterium stain GV2260 (OD600=1.0) carrying silencing constructs were infiltrated into 2 fully expended leaves for inducing gene silencing.  Samples were collected at 8, 10, 12, 14, and 16 days post Agro-inoculation (DPI) for RPN9-silenced plants and N-silenced control. For RPN8-silencing, samples were collected at 8, 10, and 12 DPI, and the empty vector treated plants were used as a control. Each sample was a pool of 6 silenced leaves collected from 3 individual plants. All the samples were in biological triplicates from 3 sets of independently silenced plants. Total RNA was extracted using Trizol and DNA was removed with DNase I treatment before cDNA synthesis.
Keywords: Reference design

overall_design: 25 hybs total

first: Jin

last: Hailing


title: cDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype

city: Utrecht

first: Nagesha

middle: A S

last: Rao

state: Utrecht

department: Veterinary sciences

country: Netherlands

institute: Utrecht University

summary:
Mammary cancer is the most common type of cancer in female dogs with a lifetime risk of over 24% when dogs are not spayed. The elucidation of the complete canine genome opens new areas for development of cancer therapies. These should be tested first by in vitro models such as cell lines. However, to date, no canine mammary cell lines have been characterized by expression profiling. In this study, canine mammary tumour cell lines with histologically distinct primary tumours of origin were characterized using a newly developed canine cDNA microarray. Comparisons of gene expression profiles showed enrichment for distinct biological pathways and were related to biological properties of the cell lines such as growth rate and in vitro tumourigenicity. Additionally, gene expression profiles of cell lines also showed correspondence to their tumour of origin. Major differences were found in Wnt, cell cycle, cytokine/Rho-GTPase, alternative complement and integrin signalling pathways. Because these pathways show an overlap at the molecular level with those found in human breast cancer, the expression profiling of spontaneous canine mammary cancer may also function as a biological sieve to identify conserved gene expression or pathway profiles of evolutionary significance that are involved in tumourigenesis. These results are the basis for further characterization of canine mammary carcinomas and development of new therapies directed towards specific pathways. In addition these cell lines can be used to further investigate identified deregulated pathways and characterize until now unannotated genes.
Keywords: cell line type comparision

overall_design: Three canine mammary tumor cell lines (CMT) originating from benign mixed tumor (CMT-U229), primary mammary osteosarcoma (CMT-U335) and primary mammary anaplastic carcinoma (P114) were compared directly to each other in this study. Total RNA was isolated from cells grown to near confluence. In vitro transcription followed by labeling and hybridization to cDNA microarray was carried out. In a loop design of hybridization, labeled cRNA from cell lines were hybridized against each other.  Statistical analysis of the log transformed normalized data was done using SAM (significance analysis of microarray) and differentially expressed genes from each experimental subset (comparison of two cell lines) were identified. Dye swaps and at least one biological replicates were included under each experimental subset (each comparision).

middle: A

first: Monique

last: van Wolferen

first: René

last: van den Ham

first: Dik

last: van Leenen

first: Marian

last: Groot Koerkamp

first: Frank

last: Holstege

first: Jan

last: Mol


title: Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes_Liver

status: Public on Aug 29 2007

city: Vienna

first: J. Andrew

last: Pospisilik

address: Dr. Bohr-gasse 3

country: Austria

institute: IMBA

summary:
Transcriptional profiling of muscle and liver samples from muscle- and liver-specific AIF knockout mice respectively. Samples obtained from 8 week old, male knockouts, backcrossed 8 times onto the C57Bl6 background are compared to those obtained from littermate control mice.
Keywords: disease state analysis, AIF knockout, metabolism

overall_design: For the muscle experiment single samples from three different knockout mice are compared with samples fom three independent control mice. For the liver experiment samples are obtained from 4 liver AIF knockouts and compared with a pooled sample obtained from 4 different control mice.

first: J

first: Josef

last: Penninger


title: Transcriptome profiling of H. polymorpha in response to Cd exposure.

status: Public on Aug 30 2007

city: Daejeon

first: Min Jeong

last: Sohn

address: 52 Eoeun-dong, Yuseong-gu

department: Protein therapeutics research center

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology

summary:
To gain insight into the basic mechanism of Cd detoxification in the methylotrophic yeast, H. polymorpha, we analyzed temporal changes in transcriptional profiles in response to Cd exposure. We used H. polymorpha whole-genome cDNA microarrays, which contain 98% of all H. polymorpha ORFs in duplicate or triplicate.
Keywords: time course, cadmium

overall_design: Total RNA samples were collected from H. polymorpha cells after 15, 30, 60, and 120 min incubation with 300μM Cd. Using the RNA sample obtained prior to Cd addition as a reference, the differential fluorescence intensities of each RNA sample prepared at the indicated times were measured after labeling with Cy3 or Cy5 fluorochromes. For all analyses, we performed dye swapping experiments to avoid dye bias, thus four or six intensity values were generated for each ORF and averaged for analysis.

first: MinJeong

first: Jeong-Nam

last: Park

first: Doo-Byoung

last: Oh

first: Ohsuk

last: Kwon

first: SangKi

last: Rhee

first: Chul-Gu

last: Hur

first: SangYup

last: Lee

first: Gerd

last: Gellissen

first: HyunAh

last: Kang


title: Increased Susceptibility of Aging Kidney to Ischemic Injury:  Identification of Molecular Pathways using Microarray

city: College Station

first: Alan

middle: R.

last: Parrish

address: 364 Reynolds

department: Systems Biology

institute: Texas A&M Health Science Center

summary:
Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism(s) underlying the increased susceptibility to injury remain undefined. These experiments were designed to investigate the influence of age on the response of the kidney to ischemic and nephrotoxic challenge.   Renal slices were prepared from young (4 month), aged ad libitum (aged-AL; 24 month) and aged caloric restricted (aged-CR; 24 month) male Fischer 344 rats and subjected to ischemic stress (anoxia, 100% N2) for 0-60 min and to cisplatin (2 mM, 4hr) challenge.  As assessed by biochemical and histological evaluation, slices from aged-AL rats were more susceptible to injury than young counterparts.   Importantly, caloric restriction attenuated the increased susceptibility to injury.  In an attempt to identify the molecular pathway(s) underlying this response, microarray analysis was performed on tissue harvested from the same animals used for the functional analysis.  RNA was isolated and the corresponding cDNA was hybridized to CodeLink™ Rat Whole Genome Bioarray slides.  Subsequent gene expression analysis was performed using GeneSpring software.  Using two-sample t-tests and a 2-fold cut-off, the expression of 92 genes was changed during aging and attenuated by caloric restriction.  In summary, several changes were identified that may be associated with the increased susceptibility of aging kidney to injury.
Keywords: aging, caloric-restriction, kidney

overall_design: This study utilized three groups (young, aged ad libitum, and aged caloric-restricted) each with 4 biological replicates for a total of 12 samples.  Each sample was hybridized to a single one-color array platform with no reference samples being used for a total of 12 arrays.

first: Gang

last: Chen

first: Eric

last: Bridenbaugh

first: David

last: Zawieja

first: Robert

last: Burghardt

middle: R


title: Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes_Muscle

first: J. Andrew

first: J


title: Array-CGH screening of pediatric T-lymphoblastic leukemias (n=73)

status: Public on Aug 25 2007

city: Heidelberg

first: Stefan

last: Pfister

address: INF 580

department: Molecular Genetics

country: Germany

institute: German Cancer Research Center

summary:
DNA copy-number profiling of 73 T-ALLs
Keywords: Genetic modification

overall_design: Comparison of tumor DNA (tumor) versus control DNA (pool of lymphocyte DNA from 10 healthy donors). Whole-genome screening for DNA copy-number aberrations using array-based comparative genomic hybridization (aCGH). Tumor DNA labeled in Cy3 and control DNA labeled in Cy5.

first: Marc

last: Remke

first: Wiebke

last: Werft

first: Grischa

last: Toedt

first: Bernhard

last: Radlwimmer

first: Andreas

last: Kulozik

first: Peter

last: Lichter


title: Chip-chip from pro-B cells with H3K4me3, H3K4me2 and H3K9ac

status: Public on Aug 23 2007

first: Meinrad

last: Busslinger

address: Dr. Bohr-Gasse 7

institute: Research Institute of Molecular Pathology

type: Genome binding/occupancy profiling by genome tiling array

summary:
The transcription factor Pax5 represses B-lineage-inappropriate genes and activates B-cell-specific genes in B-lymphocytes. Here we have identified 170 novel Pax5-activated genes. Conditional mutagenesis demonstrated that the Pax5-regulated genes require continuous Pax5 activity for normal expression in pro-B and mature B cells. Expression of half of the Pax5-activated genes is either absent or significantly reduced upon Pax5 loss in plasma cells. Direct Pax5 target genes were identified based on their protein synthesis-independent activation by a Pax5-estrogen receptor fusion protein. Chromatin immunoprecipitation (ChIP) of Pax5 together with chromatin profiling by ChIP-on-chip analysis demonstrated that Pax5 directly activates the chromatin at promoters or putative enhancers of Pax5 target genes. The novel Pax5-activated genes code for key regulatory and structural proteins involved in B cell signaling, adhesion, migration, antigen presentation and germinal center B cell formation, thus revealing a complex regulatory network, which is activated by Pax5 to control B cell development and function.
Keywords: Chip-chip, cell type comparison

overall_design: comparison of Pax5-/-Rag2-/- vs Rag2-/- pro-B cells

first: Shane

last: McManus


title: ANALYSIS OF GENE EXPRESSION IN EARLY-STAGE OVARIAN CANCER (1)

city: Milan

first: Pietro

last: Mariani

address: Via La Masa 19

country: Italy

institute: Mario Negri Institute

summary:
Gene expression was analysed in 68 stage I ovarian cancers and 15 borderline samples using microarrays. Tumors were obtained directly at surgery and immediately frozen in liquid nitrogen until analysis. Glass arrays containing 16000 genes were used. Data was interpreted by unsupervised and supervised analysis. All tumors had a wild-type p53. Unsupervised analysis identified eight major clusters one of which was statistically associated to overall survival, grading and histotype (cluster C) and another with grading and histotype (cluster D). Supervised analysis showed a clear association between histotypes and gene expression profile and distinguished the four major histotypes (serous, clear cells, mucinous, endometrioid). The data also discriminated between the different grades of the tumors. The subset of 15 borderline tumors were undistinguishable from grade 1 tumors. When patients, relapsing or not, were analysed, a subset of genes able to differentiate them was identified. The genes identified in the major clusters belong to general classes of lipid biosynthesis, transport and metabolism, immunoglobulins (antigen presenting and processing) and genes acting as metal, ion and anion transporters including ATPase activity coupled with ion transport.
Keywords: disease state analysis

overall_design:
Gene expression for 83 ovarian tumor samples was evaluated: 68 from patiens presenting stage I carcinoma and 15 with borderline desease.
A common reference design was applied and each samples was cohybridizated with a universal reference.
Also a dye swap control has been done so that for the two technical replicates of each sample an expression profile of raw data was obtained.
These data were independently processed and combiend to have a unique normalized profile for each sample.


title: ANALYSIS OF GENE EXPRESSION IN EARLY-STAGE OVARIAN CANCER (2)


title: Hybridization of four Euphorbs on Euphorbiaceae arrays

status: Public on Aug 24 2007

city: Fargo

last: Horvath

state: ND

address: 1605 Albrecht Blvd

department: Agr. Research Service

institute: US Dept. Agriculture

summary:
These experiments were designed to determine the usefulness of the spurge/cassava euphorbiaceae arrays for investigating gene expression in other euphorbs.
Keywords: heterologous hybridization euphorbia

sample_organism:
Manihot esculenta
Ricinus communis
Euphorbia esula
Euphorbia pulcherrima

platform_organism:
Manihot esculenta
Euphorbia esula

overall_design: two color dye swap design

middle: P

first: James

middle: V

last: Anderson


title: Inhibition of Respiration by Nitric Oxide Induces a Mycobacterium tuberculosis Dormancy Program

status: Public on Aug 22 2007

city: Stanford

first: SMD

last: Staff

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

summary: An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis.

overall_design: The quality controls were biological replicate and technical replicate

first: Martin

middle: I

last: Voskuil

first: Dirk

last: Schnappinger

first: Kevin

last: Visconti


title: Expression data from leaf of Arabidopsis TGS mutants

city: Tsukuba

first: Yoshiki

last: Habu

state: Ibaraki

address: Kannondai 2-1-2

department: Institute of Agrobiological Sciences

institute: National Agriculture and Food Research Organization

summary:
Transcriptional gene silencing (TGS) in Arabidopsis is regulated by two distinct pathways, DNA methylation-dependent and –independent. ddm1 and mom1 mutants are defective in the DNA-dependent and –independent pathway, respectively.
We used microarrays to detail the global changes in gene expression in the TGS mutants
Keywords: TGS mutants

overall_design: We sought to obtain differential gene expression in each mutant in order to characterize the effect of deficiency in two distinct TGS pathways. To that end, we took mutants that regulate TGS by DNA methylation-dependent (ddm1) and -independent (mom1) pathways, respectively. Total RNA was extracted from leaves of these mutants and subjected to hybridization on Affymetrix Arabidopsis 8K microarrays.

first: Jerzy

last: Paszkowski

first: Tong

last: Zhu


title: Plant genes affected by Solanum tuberosum and Colorado potato beetle interaction

city: Rockville

first: Jia

last: Liu

address: 9712 Medical Center Drive

institute: Plant Genomics

summary:
Potato leaves From Solanum tuberosum var. Kennebec will be wounded and oral secretions from 4th instar CPB will be isolated and added to the plants as described by Kruzmane et al (2002, Physiol. Plantarum 115:577-584).  The leaf from the 6th node of the potato plant will be wounded or wounded and treated with oral secretions from CPB. Unwounded leaves from node 1-5 of the wounded  and wounded plus oral secretions plants will be harvested as systemic material. The leaves will be harvested after 4 hrs and RNA will be isolated. 4 hrs was chosen because this represents a time when early and late induced genes should both be present. In addition, the leaf from the 6th node will be subjected to feeding by CPB that have been raised on potato leaves and starved for 16 hrs immediately prior to infestation. Insects will be allowed to feed for 1 hr and the leaves will be harvested after 3 additional hrs. An additional set of plants will be used to infest the leaf on the 6th node for 4 hrs. Leaves from the 6th node will be collected from uninfested plants after 4 hrs as a control. Three sets of 6-12 plants will be used for each sample.
Keywords: Direct comparison

overall_design: 24 hybs total

first: Lawrence

last: Susan


title: Normal colon top vs colon crypts

status: Public on Aug 28 2007

city: San Francisco

first: Xin

last: Chen

institute: UCSF

summary:
The study is to define the genetic programs of human colonic cell differentiation, and to identify candidate markers for colonic stem cells and stem cell niche
The study assayed the expression of 9 paired normal colon top versus crypts.
Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Keywords: Logical Set

overall_design: Using regression correlation


title: Genome-wide gene expression in response to mating pheromones in S. pombe

city: Huddinge

first: Anthony

last: Wright

address: Blickagången 6

institute: Karolinska Institute

summary:
Fission yeast cells undergo sexual differentiation in response to nitrogen starvation. In this process haploid M and P cells first mate to form diploid zygotes, which subsequently enter meiosis and sporulate. Prior to mating, M cells secrete M-factor and P-cells secrete P-factor, and these diffusible mating pheromone can activate a signal transduction pathway in the opposite cell type. The pheromone response orchestrates mating and is also required for entry into meiosis. Here we use DNA microarrays to identify genes that are induced by M-factor in P cells and by P-factor in M-cells. The use of a cyr1 genetic background allowed us to study pheromone stimulation independently of nitrogen starvation. We identified a total of 163 genes that were consistently induced more than two-fold by pheromone stimulation. As expected, there was a substantial overlap between the genes induced by M- and P-factor. Surprisingly, we found that pheromone control extended to genes fulfilling their function well beyond the point of entry into meiosis, including numerous genes required for meiotic recombination. A direct comparison of the M- and P-factor induced expression pattern allowed us to identify cell-type specific transcripts, including three new M-specific genes and one new P-specific gene. Finally, our results support the notion that Ste11 is the key transcription factor activated by pheromone signalling.
Keywords: dose response

overall_design: Gene expression profiling experiment in which gene expression in pheromone treated cultures was compared to that of untreated control cells. All cultures were growing exponentially in MSL minimal medium. S. pombe h+ cyr1- cells treated with M-factor mating pheromone for 2, 4 and 6 hrs respectively, were compared to non-treated cells. Similarly, S. pombe h- sax2- cyr1- cells treated with P-factor mating pheromone for 2, 4 and 6 hrs, were compared to non-treated cells. For identiofication of cell-type specific gene expression, h+ cyr1- cells were compaired  to h+ sxa2- cyr1- cells. This was done both before pheromone treatment (0h/0h) and after 6 hours of pheromone treatment (6h/6h). RNA was extracted and subjected to cDNA expression profiling analysis using the established protocols (Xue et al, Yeast 21, 25-39, 2004).Data normalized with 'Lowess' per chip per spot normalization.

first: Yongtao

last: Xue-Franzen

first: Soren

last: Kjarulff

first: Olaf

last: Nielsen


title: Nicotiana nectary development

summary:
Floral Nectaries Many plants secrete a rich floral nectar to entice visitation by insect and avian pollinators.  In turn, these pollinators transfer pollen between flowers increasing plant fecundity.  The nectary is the floral organ that secretes nectar into the base of the flower.  The size and abundance of the ornamental tobacco nectaries (Nicotiana sp.) will permit us to isolate up to several grams of nectaries at each stage to obtain the necessary amounts of RNA for probe preparation.  Our primary goals to understand the biochemistry the nectary, so that we can manipulate nectary function to increase pollinator visitation. We have previously conducted an EST study and have identified 13596 cDNAs from three different stages of nectary development (Stage 6, immature, presecretory nectaries; Stage 12, mature nectaries at floral anthesis; and nectaries, 44 hours after fertilization.  In our efforts to evaluate the transcriptional program for the Nicotiana nectary we are proposing to evaluate nectary mRNAs by hybridization with the potato microarrays.    We have preliminary evidence that wholesale transcriptional reprogramming (60% of the transcriptome) occurs during nectary maturation and again following fertilization. Our goal is to understand these processes at a biochemical level so that we can begin manipulating nectary function to improve nectar quality and quantity thereby increasing the attractiveness of flowers to insect pollinators.  Such improvements have the potential to result in increases in insect visitation, seedset, and ultimately yield for insect pollinated crops.  We are also making significant efforts to understand the restructuring of the nectary during its lifecycle. Many changes occur during nectary development and the observed transcriptional reprogramming makes sense the when these many changes are accounted for.
Keywords: Loop design

sample_organism: Nicotiana langsdorffii x Nicotiana sanderae

overall_design: 30 hybs total

first: Thornburg

last: Robert


title: A member of the cAMP receptor protein family of transcription regulators in M. tuberculosis is required for virulence

status: Public on Aug 21 2007

summary:
Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow-derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.
Keywords: strain or line design, genetic modification design, and comparative genome hybridization design

overall_design: Six samples were analyzed. The quality controls were biological replicate and technical replicate and dye swap quality control

first: Lisa

last: Rickman

first: Colin

last: Scott


title: methylatedDNA_peripheralBlood

first: Jiexin

last: Zhang

address: 1515 Holcombe Blvd

department: Bioinformatics & Computational Biology

institute: UT MD Anderson Cancer Center

type: Methylation profiling by genome tiling array

summary:
The goal of the experiment – genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters
Keywords: DNA methylation, Methylated CpG island amplification coupled with promoter arrays, normal tissue

overall_design: DNA methylation was initially suggested to control tissue-specific gene expression in mammalian cells, but CpG islands (CGIs) were found to be generally free of methylation regardless of the expression status of the associated genes. Here, we have discovered an exceptional subset of autosomal genes associated with dense promoter CGIs (4.1% of all CGIs analyzed) that is methylated in normal tissues.  We observed tissue-specific gene silencing for this class of genes, and provided evidence for a direct role of methylation in maintaining the silencing state via pharmacologic or genetic reduction in DNA methyltransferase activity.  By comparing DNA sequences flanking the center of CGIs, we have identified five sequence motifs significantly enriched in genes with methylated CGIs, suggesting the influence of cis regulatory elements on the establishment and/or stability of DNA methylation.  Together, these results provide important new insights into the role of CGI methylation in normal development and differentiation.

first: Lanlan

last: Shen

first: Yutaka

last: Kondo

first: Yi

last: Guo

first: Li

first: Saira

last: Ahmed

first: Jingmin

last: Shu

first: Xinli

first: Robert

last: Waterland

first: Jean-Pierre

last: Issa


title: Colon tumor samples

status: Public on Aug 17 2007

city: Stockholm

first: Annika

last: Saaf

department: Department of Molecular Medicine & Surgery

institute: Karolinska Institute, Karolinska University Hospital, Solna

summary:
In response to polarization cues, cultured Caco-2 cells, a human colon adenocarcinoma-derived cell line, form a polarized epithelium resembling normal enterocytes.  We investigated potential signaling mechanisms activated by Caco-2 cells that might trigger the genome-wide transcriptional reprogramming that accompanies polarization (Saaf et al, submitted-I). cDNA microarrays were used to compare the transcriptional profile of Caco-2 polarization to the gene expression profiles of normal human colon and colon tumors. The transcript profile of proliferating, non-polarized Caco-2 cells has striking parallels to the gene expression profile of human colon cancer in vivo. However, as Caco-2 cells develop polarity, the gene expression profile shifts to one more closely resembling that of normal colon tissue, suggesting that the underlying regulatory mechanisms that mediate Caco-2 cell polarization are similar to those that occur during in vivo enterocyte differentiation. We show that transcriptional re-programming of Caco-2 cells during development of cell polarity occurs in the context of signaling pathways that are regulated in a manner that is remarkably similar to those in normal intestinal development. For example, transcriptional targets of the Wnt pathway are tightly regulated during Caco-2 cell polarization, mimicking the gradient of Wnt-mediated transcription in the crypt (high expression) to villus (low expression) axis in human intestine. However, Caco-2 cells lack full-length APC necessary for normal Wnt-regulated degradation of beta-catenin. Biochemical analysis indicates that regulation of the Wnt pathway occurs in the nucleus at the level of activation of target genes by the beta-catenin-TCF complex, revealing a novel additional mechanism by which intestinal cells may regulate Wnt signaling during their maturation. In addition, other signaling pathways including Notch, BMP, Hedgehog, and growth factor, were temporally regulated during Caco-2 cell polarization. Surprisingly, modulation of these signaling pathways in Caco-2 cells occurs in the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ.  This dataset contains gene expression profiles of 9 normal colon samples and 15 colon tumor samples.  Samples of tumor and normal colon mucosa were collected from colon cancer resection from Department of Surgery, Queen Mary Hospital University of Hong Kong. Tissue was frozen in liquid nitrogen within 30 min of resection. Nonneoplastic mucosa from colon was dissected free of muscle and histologically confirmed to be tumor free by frozen section. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) from each tissue sample and processed for microarray hybridization.
A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied.
Disease State: Tumor/Normal colon samples
Keywords: disease_state_design

first: Xin


title: Gene Expression Profiling of Patients with a Variation in the TNF-alpha gene and suffering from Multiple Trauma

city: Giessen

first: Hamid

last: Hossain

address: Frankfurter Str. 107

department: University of Giessen

institute: Institute for Medical Microbiology

summary:
BACKGROUND:
Patients encountering severe trauma are at risk of developing sepsis syndrome and subsequent multiple organ failure. This is often associated with fatal outcome despite survival of the initial injury. We undertook a prospective cohort study of trauma patients to examine the role of tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of sepsis syndrome and mortality.

METHODS:
159 severely traumatized patients from a single centre were included. Serial blood samples were analyzed for serum concentrations of TNF-alpha and lymphotoxin alpha (LT-alpha). We genotyped nine polymorphisms in the TNF gene and tested for an association with sepsis syndrome and outcome. Genetic associations were validated in an external replication sample (n=76). We examined the peripheral blood transcriptome in n=28 patients by whole genome-based profiling and validated the results.

RESULTS:
Carriage of the TNF rs1800629 A allele was associated with higher TNF-alpha serum concentrations on the first day after trauma and during follow up (two-sided p=5.0x10-5), with development of sepsis syndrome (OR 7.14, two-sided p=1.2x10-6; external validation sample (n=76): OR 3.3, one-sided p=0.03), and with fatal outcome (OR 7.65, two-sided p=1.9x10-6). Carriage of the TNF rs1800629 A allele was associated with differential expression of genes representing stronger pro-inflammatory and apoptotic responses as compared to carriage of the wild type allele.

CONCLUSIONS:
Common TNF gene variants are associated with sepsis syndrome and death after severe injury. These findings are strongly supported by functional data and may be important for developing preemptive anti-inflammatory interventions in carriers of the risk-associated allele.
Keywords: Disease state analysis

overall_design:
159 consecutive traumatized patients were included in the study upon admission to the intensive care unit (ICU), fulfilling all of the following inclusion criteria: severe injuries of at least two body regions or three major fractures, between 18 and 65 years of age, an estimated Injury Severity Score (ISS)21 of ≥12 points after thorough assessment of injuries, <12 hours between the occurrence of the accident and time of admission to the ICU, and at least more than 3 days of survival. None of the patients underwent neuro- or cardiac surgery. We excluded patients with severe intracranial head injuries, coagulation abnormalities known at the day of admission to the ICU, acute renal failure, liver failure, malignant disease, or hemofiltration in the patient`s history. All patients were genotyped for nine polymorphisms in the TNF gene and tested for an association with sepsis syndrome and outcome. The genotyping results revealed significant association for the carriage of a TNF rs1800629 A allele with higher TNF-alpha serum concentrations on the first day after trauma and during follow up, with development of sepsis syndrome (OR 7.14), and with fatal outcome (OR 7.65). To obtain more insight into the biological mechanisms underlying the findings in the genotyping study, we searched for differences in the peripheral blood transcriptome from patients with and without the TNF rs1800629 A variant. From 28 patients, we examined the transcpriptome from peripheral blood using the CodeLink UniSet Human 10 K Bioarrays (GE Healthcare, Freiburg, Germany). Only whole blood samples drawn upon admission to the ICU were used for this investigation. Microarray results were validated by quantification of mRNA by TaqMan® technology. The patients were dichotomized into two groups: group A) 16 patients  without the TNF rs1800629 A variant (WT1-16) and
group B) 12 patients  carrying the TNF rs1800629 A variant (MUT1-12).
Each patient sample was hybridized in duplicate on microarrays (label-extract technical replicate). A total of 75 arrays (42 group A, 33 group B) were subjected to microarray quality analysis. Of these, 17 arrays (12 group A, 5 group B) were excluded from the data set due to quality reasons and a final set of 58 arrays (30 group A and 28 group B) was subjected to microarray analysis. The arrays were designated WTx.y or MUTx.y with WT for patients with and MUT for patients without the TNF rs1800629 A variant, with x for the patient-ID (1,2,3,4..) and y for the technical replicate number (1,2 or 3).

first: Trinad

last: Chakraborty

first: Thilo

last: Menges

first: Gregor

last: Bein

first: Inke

last: Koenig

first: Simon

last: Little

first: Svetlin

last: Tchatalbachev


title: Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis

first: SMD

last: Staff

summary:
Unlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha-crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.
Keywords: strain or line design, genetic modification design and comparative genome hybridization design

overall_design: 12 samples were analyzed. The quality controls were biological replicate and technical replicate

first: Heui-Dong

last: Park

first: Martin

last: Voskuil


title: Opaque vs Transparent transcriptome of Streptococcus pneumoniae serotype 2 strain D39

city: Adelaide

first: Uwe

last: Stroeher

state: South Australia

address: North Terrace

department: School of Molecular and Biomedical Science

institute: University of Adelaide

summary:
Invasive pneumococcal disease is preceded by asymptomatic colonization of the human nasopharynx by Streptococcus pneumoniae. Progression from colonization to invasion is a watershed in the host-pathogen interaction, and exposes the pneumococcus to markedly different microenvironments. This in turn, requires alterations in gene expression profile to adapt to the new niche. One apparent adaptive mechanism is reversible phase variation between “transparent” and “opaque” colony opacity phenotypes. Transparent phase variants colonize the nasopharynx more efficiently than opaque variants of the same strain, while opaque variants exhibit higher systemic virulence. Previous studies have reported quantitative differences in surface components such as the capsule, teichoic acid and certain surface proteins between the two phenotypes, but the underlying regulatory mechanism is not understood. In the present study, we found no differences in expression of key surface proteins between opaque and transparent variants of S. pneumoniae strain D39, but opaque cells produced five-fold more capsular polysaccharide. Subsequent microarray and real-time RT-PCR analysis showed no differences in capsule gene expression, but several genes involved in uridine monophosphate (UMP) biosynthesis were up-regulated in the opaque phenotype.  This correlated with significant increases in the intracellular concentrations of both UMP and UDP-glucose, which are essential precursors for capsule biosynthesis. Our data suggest a novel mechanism for pneumococcal capsule regulation, in which rate-limiting precursor pathways are modulated rather than the capsule biosynthetic genes themselves.
Keywords: Phase variants

overall_design: One condition experiment examining the transcriptome between two phases of the same organsism

middle: H

first: James

last: Paton


title: Caco2 polarization time-course

summary:
CELL CULTURE: The human intestinal epithelial cell line Caco-2 (obtained from Dr. Stanley Falkow, Stanford University) was used to study the genome-wide expression program underlying the transition from cell proliferation to establishment of a polarized epithelial layer. Cells were first grown in sparse culture on plastic p15 dishes to generate a population of "contact naive" cells.  These cells were combined and plated at confluency on Costar filter inserts. The day cells were plated on filters is designated the zero time-point. Cells were cultured for 26 days, during which time they develop structural and functional polarity. Media was changed every other day. Triplicate samples were harvested at eleven time-points over the time course for microarray analysis. The entire time course was performed twice.  Three control arrays are included in this data set; (1) to determine how the gene-expression patterns in Caco-2 cells grown on a plastic dish compares to the expression profiles identified in Caco-2 cells grown on Costar filter inserts (microarray shcu182, data not shown), (2) to tested whether feeding the cells at different time-points had an effect on the Caco-2 gene-expression program.  All samples taken (except at the day zero time-point) were fed with fresh media one day prior to harvesting the sample. During TC3 and TC4, we performed two test arrays analyzing the transcriptional program in Caco-2 cells that were fed with fresh cell culture media 4h prior to taking the 7D time-point (microarrays shcu177 and shcu178, indicated with * in table S1).
Groups of assays that are related as part of a time series.
See additional information on overall design in Series supplementary file.

ABSTRACT: Although there is considerable evidence implicating post-translational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell-cell adhesion-initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts between neighboring cells. Expression of a cluster of genes involved in cell proliferation switched off and concomitantly the expression of genes related to functional specializations of polarized epithelial cells was induced. Induction of these latter genes correlated with formation of important structural and functional features related to enterocyte differentiation and establishment of structural and functional cell polarity. Coordinated expression of genes encoding components of functional cell structures were often observed, indicating temporal control of expression and assembly of multiprotein complexes. Changes in gene expression patterns during Caco-2 cell differentiation in vitro paralleled those in terminally differentiating enterocytes migrating up the intestinal crypt-villus axis in vivo, despite the lack of stromal cells and gradients of morphogens in this tissue culture model.
Keywords: time_series_design

overall_design: Computed


title: Genomic comparison in Enteroheamorragic E. coli O157 and non-O157 serotypes by comparative genomic hybridization.

city: Kiyotake

first: Yoshitoshi

last: Ogura

state: Miyazaki

address: Kihara 5200

department: Frontier science reserch center

institute: University of Miyazaki

summary:
Background: Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-bone illness in humans. The chromosome of O157 consists of 4.1-Mb backbone sequences shared by benign E. coli K-12, and 1.4-Mb O157-specific sequences encoding many virulence determinants such as Shiga toxin genes (stxs) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to clonal lineages distinct from O157 also cause similar illness in humans. According to the parallel evolution model, they have independently acquired the major virulence determinants, stxs and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet systematically been analyzed.     Results: By using the microarray and Whole Genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Only around 20% of the O157-specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes found on prophages and plasmids in O157, and also multiple prophages similar but significantly divergent from those in O157.  Conclusion: Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants appear to be involved in the parallel evolution of EHEC.
Keywords: comparative genomic hybridization, CGH

overall_design: Total 29 test samples were analyzed. Genomic DNA from each test strain and a reference strain (O157 Sakai) were labeled with Cy3 and Cy5, respectively, and were cohybridized on a single array. Labeling and hybridization were performed twice independently.

first: Tetsuya

last: Hayashi


title: Ultraconserved elements are associated with control of splicing regulators by alt splicing and nonsense mediated decay

city: Santa Cruz

first: Manny

last: Ares

address: 1125 High St

department: Molecular and Cellular Biology

institute: UCSC

summary:
We blocked nonsense-mediated mRNA decay (NMD) pathway and used a custom alternative transcript sensitive Affymetrix microarray to seek alternatively spliced RNAs whose levels increased compared to controls.
Keywords: Nonsense Mediated Decay (NMD)

overall_design: We used two difference methods of NMD inhibition. In one method, we transfected N2A cells with Upf1 siRNA. In the second method, we treated N2A with emetine. Three individual total RNA samples from Upf1 siRNA transfected cells and control siRNA transfected cells, and two individual total RNA samples from each of emetine treated cells and control cells, were applied on custom Affymetrix microarrays with probes specific to different alternative splicing events, including splice junction probes. Perfect match probes only.

first: Julie

middle: Z

last: Ni

first: Leslie

last: Grate

first: Christi

last: Preston

first: Naomi

last: Nobida

first: Georgann

last: OBrien

first: Tyson

last: Clark

first: John

last: Blume


title: Map transformed DNA into W6G from VCXB21

status: Public on Aug 16 2007

last: Stanford Microarray Database

summary:
Vibrio cholerae isolate W6G transformed with DNA from VCXB21 and selected for chitosan metabolism.  Nine independent transformants compared to parental W6G on microarrays containing probes for all N16961 ORFs.  Regions acquired from VCXB21 hybridize more strongly to the array.
A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's.
Keywords: genotyping_design

first: Michael

last: Miller


title: Gene expression profiling in three primary human cell lines treated with atorvastatin or an inhibitor of Rho kinase

city: Little Rock

first: Marjan

last: Boerma

state: AR

address: 4301 West Markham, Slot 725

institute: University of Arkansas for Medical Sciences

summary:
Inhibitors of Rho kinase (ROCK) are a new class of drugs with potential benefit in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK inhibitors modulate many cellular functions, some of which are similar to the pleiotropic (non-lipid lowering) effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds which inhibit both isoforms of ROCK, ROCK1 and ROCK2. In this study, the effect of the newly developed ROCK2 inhibitor SLx-2119 on genome-wide gene expression was compared with atorvastatin in primary human cells.
Keywords: Cell type comparison, agent comparison

overall_design:
Primary cultures of human microvascular endothelial cells HMVEC), human pulmonary artery smooth muscle cells (PASMC), and normal human dermal fibroblasts (NHDF) were treated with SLx-2119 or atorvastatin for 24 hours, after which total RNA was isolated and gene expression profiles were obtained with Illumina BeadChips.
Reversing the effects of statins by the addition of mevalonate provides an opportunity to subtract non-specific effects of statins in vitro. This approach was also used in the current study, as follows: cells were treated with a combination of atorvastatin and mevalonate and genes were considered up- or down-regulated when the addition of mevalonate significantly reversed the change in expression observed in atorvastin-treated cells.

first: David

middle: S

last: Loose

last: Hauer-Jensen


title: FIGLA, an oocyte-specific transcription factor, affects ovarian gene expression

city: bethesda

first: saurabh

last: joshi

address: 9000 rockville pk

department: national institute of diabestes digestive and kidney diseases

institute: national institutes of health

summary:
Ovarian folliculogenesis in mammals is a complex process involving cross talk between germ and somatic cells. Carefully orchestrated expression of transcription factors, cell adhesion molecules and growth factors are required for success. We have identified a germ-cell specific basic helix-loop-helix transcription factor, FIGLA (Factor In GermLine, Alpha) and demonstrated its involvement in two independent developmental processes: formation of primordial follicle and coordinate expression of zona pellucida genes. Taking advantage of Figla null mouse line, we have used microarrays to identify potential downstream target genes. Using high stringent cut offs, we find that FIGLA functions a key regulatory molecule in coordinating expression of the NALP family of genes, genes of known oocyte-specific expression and a set of functionally un-annotated genes. These data implicate FIGLA as a central regulator of oocyte-specific genes that play role in folliculogenesis and early development.
Keywords: Time Course; genetic modification

overall_design: We analyzed 8 groups of arrays, 26 arrays total. Four arrays of WT newborn ovary RNA (cy5) versus KO newborn ovary RNA (Cy3), then four arrays of the same samples dye-reversed. Three arrays of WT E17.5 ovary RNA (cy5) versus KO E17.5 ovary RNA (Cy3), then three arrays of the same samples dye-reversed. Three arrays of WT E14.5 ovary RNA (cy5) versus KO E14.5 ovary RNA (Cy3), then three arrays of the same samples dye-reversed. Three arrays of WT E12.5 ovary RNA (cy5) versus KO E12.5 ovary RNA (Cy3), then three arrays of the same samples dye-reversed.

first: Saurabh

last: Joshi

first: Lauren

last: Sims

first: Shawn

last: Levy

first: Jurrien

last: Dean


title: Comparison of RNA Amplification Techniques meeting the demands for the Expression Profiling of Clinical Cancer Samples

city: Seibersdorf

first: Martin

last: Lauss

state: Lower Austria

address: Mendelstrasse 1

department: Life Sciences

institute: ARC Seibersdorf research

summary:
For microarray experiments starting with nanogram amounts of RNA it is essential to implement reproducible and powerful RNA amplification techniques. Available methods were mainly tested for reproducibility, only a few studies concentrated on potential amplification bias. We evaluated three amplification protocols, which are less time-consuming than the commonly used T7-RNA polymerase based in vitro transcription protocols and therefore may be more suitable for clinical use: Template Switching (TS)-PCR (SMART-PCR Kit, BD), Ribo-SPIA (single primer isothermal amplification, Oviation, Nugen) and a random primer-based PCR. Additionally a more sensitive labeling method, Dendrimer-labeling (Genisphere), was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. Hybridizations  were carried out on a targeted two-colour oligonucleotide microarray. From our results we conclude that RNA amplification with TS-PCR is highly reproducible and results in a reliable representation of the starting RNA population. We then assessed whether RNA amplification of clinical breast and thyroid cancer samples with TS-PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. According to our experiments TS-PCR proved to be a very reliable method for global RNA amplification, even when starting from partially degraded RNA down to a RNA Integrity Number (RIN) of 4.3.
Keywords: microarray expression profiling, RNA amplification techniques, RNA integrity

overall_design:
To compare the methods under test we generated two pools of total RNA from breast cancer cell lines MCF7 and Hs578T, respectively, which were used for to test subsequent techniques. For each method a replication including a dye-swap hybridization was performed. Over all, non-amplifying methods were replicated two times and amplifying methods 4 times. Next, we investigated whether Ribo-SPIA and TS-PCR are suitable for amplification of rather heterogenous clinical breast cancer samples. Total RNA from (estrogen receptor) ER- (patient number 180/03) and ER+ (patient number 254/03) breast cancer samples were chosen as targets for hybridization. As tumor material was limited, only one hybridization of unamplified RNA could be performed. For Ribo-SPIA and TS-PCR (17, 19 and 26 cycles) a replicate including a dye-swap hybridization was carried out.
To test test stability of TS-PCR with degraded samples, total RNA from both breast cancer samples was subjected to artificial degradation by heating to 50 degrees Celsius for 0, 25 and 50 minutes. Additionally, thyroid nodules from 3 different patients (604/05, 607/05 and 618/05) were disrupted into several pieces. TS-PCR was performed on total RNA from both breast cancer samples and different extracts from thyroid cancer subpieces, respectively. The amplification product was labeled with Cy3. Universal Reference RNA (Stratagene) was used as a common reference and labeled with Cy5.

first: Klemens

last: Vierlinger

first: Andreas

last: Weinhaeusel

first: Sandra

last: Szameit

first: Klaus

last: Kaserer

first: Christa

last: Noehammer


title: Heme_k2_Filtered bckg correct trans-norm

city: Wageningen

first: L.

middle: Mariela

last: Serrano

address: Nieuwe Kanaal 9A

institute: TI Food and Nutrition

summary:
We ran for each studied culture (WCFS1 NZ7608 NZ7602 and NZ7603) an independent shake flask cultivation and designed one experiment. The hybridization scheme (4 arrays) of the experiment was as follows: WCFS1_respiratory NZ7608_respiratory NZ7602_respiratory NZ7603_respiratory WCFS1_respiratory. In the experiment we studied the difference in response towards respiratory cultivation (OXYGEN HEME and VITAMIN K2) of each strain compared to wild type
Keywords: comparative genomic hybridization

overall_design: Per array two cDNA labeled targets were hybridized on custom designed L. plantarum WCFS1 11K Agilent oligo microarrays (GEO Acc. Nr. GPL4318) using the Agilent 60-mer oligo microarray processing protocol version 4.1. An oligo-microarray contained an average of 3 probes per transcript. Dried slides were scanned in the Scan Array Express (PerkinElmer Life Sciences; Packard Bioscience) for both Cy3 and Cy5 at 10 microns and a PMT Gain between 40-50%.  Spot intensity data was normalized and quantified (average intensity) using a customized grid in ImaGene (BioDiscovery, Inc.). Signal intensities of all probes were corrected against background in BioArray Software Environment (BASE). The ratio of each probe (M) was defined as log [cy5 intensity/cy3 intensity] where cy3 is the wild type. Fold change (FC) is defined as 2M. For the statistical analysis we used Linear Models for Microarray Analysis (Limma) (R/limma).  Significant transcripts were defined as transcripts with a False Discovery Rate (FDR) adjusted for the pvalues of less than 1% and FC = 1.5

first: L

first: Douwe

last: Molenaar

first: Bas

last: Teusink

first: Willem

last: de Vos

first: Eddy

last: Smid


title: AER-ANA_Filtered bkgr.correction transf-norm

summary:
We ran for each studied culture (WCFS1 NZ7608 and NZ7603) one aerobe and one anaerobe batch cultivation and designed one experiment. The hybridization scheme of the experiment (6 arrays) was as follows: WCFS1_aerobe NZ7608-aerobe NZ7603-aerobe WCFS1_aerobe and then three extra hybridizations: WCFS1_anaerobe w WCFS1_aerobe; NZ7608_anaerobe NZ7608_aerobe; and NZ7603_anaerobe NZ7603_aerobe. In the experiment we compared (1) the effect of oxygen in each studied culture and (2) the difference in response toward aerobe and anaerobe conditions of each mutant compared to the wild type
Keywords: comparative genomic hybridization

overall_design: Per array two cDNA labeled targets were hybridized on custom designed L. plantarum WCFS1 11K Agilent oligo microarrays (GEO Acc. Nr. GPL4318) using the Agilent 60-mer oligo microarray processing protocol version 4.1. An oligo-microarray contained an average of 3 probes per transcript. Dried slides were scanned in the Scan Array Express (PerkinElmer Life Sciences; Packard Bioscience) for both Cy3 and Cy5 at 10 microns and a PMT Gain between 40-50%.  Spot intensity data was normalized and quantified (average intensity) using a customized grid in ImaGene (BioDiscovery, Inc.). Signal intensities of all probes were corrected against background in BioArray Software Environment (BASE). The ratio of each probe (M) was defined as log [cy5 intensity/cy3 intensity] where cy3 is the wild type. Fold change (FC) is defined as 2M. For the statistical analysis we used Linear Models for Microarray Analysis (Limma) (R/limma).  Significant transcripts were defined as transcripts with a False Discovery Rate (FDR) adjusted for the pvalues of less than 10% and FC = 1.5


title: High resolution array-CGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer

city: London

first: Andrew

last: Teschendorff

address: 72 Huntley Street

department: UCL Cancer Institute/Institute for Women's Health

country: United Kingdom

institute: University College London

summary:
An oligo array based high-resolution analysis of copy number alterations in 171 primary breast tumors of relatively small size and low NPI, and 49 breast cancer cell-lines. Objectives of the study were to study the molecular taxonomy and the genomic aberration patterns in a breast cancer cohort representative of breast cancer demographics.
Keywords: array comparative genomic hybridisation

overall_design: 171 primary breast tumors from Nottingham City Hospital and 49 breast cancer cell-lines were hybridised with male DNA as reference.

first: Suet

last: Chin


title: Anopheles gambiae immune responses to human and rodent Plasmodium parasite species

status: Public on Aug 15 2007

city: Baltimore

first: George

last: Dimopoulos

address: 615 N. Wolfe Street

department: Molecular Microbiology and Immunology

institute: Johns Hopkins School of Public Health

summary:
Transmission of malaria is dependent on the successful completion of the Plasmodium lifecycle in the Anopheles vector. Major obstacles are encountered in the midgut tissue, where most parasites are killed by the mosquito’s immune system. In the present study, DNA microarray analyses have been used to compare Anopheles gambiae responses to invasion of the midgut epithelium by the ookinete stage of the human pathogen Plasmodium falciparum and the rodent experimental model pathogen P. berghei. Invasion by P. berghei had a more profound impact on the mosquito transcriptome, including a variety of functional gene classes, while P. falciparum elicited a broader immune response at the gene transcript level. Ingestion of human malaria-infected blood lacking invasive ookinetes also induced a variety of immune genes, including several anti-Plasmodium factors.
Keywords: Anopheles gambiae, Plasmodium falciparum, ookinete, invasion, innate immunity

overall_design:
By comparing gene expression patterns between carcassess or guts of mosquitoes that fed on a P. falciparum or P. berghei wt and mosquitoes that fed on invasion incapable strains we gain information on the A. gambiae transcriptional responses to the invading ookinete at 24 hours after feeding.

By comparing gene expression patterns between carcassess or guts of mosquitoes that fed on a P. falciparum ookinete invasion incapable strain and mosquitoes that fed on non-infected blood we gain information on the A. gambiae transcriptional responses to malaria infected blood in absence of ookinete invasion at 24 hours after feeding.

By comparing gene expression patterns between mosquitoes at 4 hours after being injected with either E. coli or S. aureus and mosquitoes injected with sterile PBS we gain information on the mosquito's transcriptional response to these bacterial challenges.

first: Yuemei

last: Dong

first: Ruth

last: Aguilar

first: Zhiyong

last: Xi

first: Emma

last: Warr

first: Emmanuel

last: Mongin


title: Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy

first: SMD

last: Staff

institute: Stanford Microarray Database (SMD)

summary:
The innate mechanisms used by Mycobacterium tuberculosis to persist during periods of non-proliferation are central to understanding the physiology of the bacilli during latent disease. We have used whole genome expression profiling to expose adaptive mechanisms initiated by M. tuberculosis in two common models of M. tuberculosis non-proliferation. The first of these models was a standard growth curve in which gene expression changes were followed from exponential growth through the transition to stationary phase. In the second model, we followed the adaptive process of M. tuberculosis during transition from aerobic growth to a state of anaerobic non-replicating persistence. The most striking finding from these experiments was the strong induction of the entire DosR dormancy regulon over approximately 20 days during the long transition to an anaerobic state. This is contrasted by the muted overall response to aerated stationary phase with only a partial dormancy regulon response. From the results presented here we conclude that the respiration-limited environment of the oxygen-depleted NRP model recreates at least one fundamental factor for which the genome of M. tuberculosis encodes a decisive adaptive program.
Keywords: comparative genome hybridization design and stimulus or stress design

overall_design: 55 samples were analyzed. The quality controls were biological replicate and technical replicate

first: M

last: Voskuil

first: K

last: Visconti