title: Global gene expresssion profiling the gills of amoebic gill disease (AGD) affected Atlantic salmon (Salmo salar)

status: Public on Nov 15 2007

city: Victoria

first: Ben

last: Koop

address: PO Box 3020 STN CSC

institute: The University of Victoria

summary:
Amoebic gill disease (AGD) is an ectoparasitic condition of some farm-reared marine fish and is caused by Neoparamoeba perurans. Tanks housing Atlantic salmon (Salmo salar) were inoculated with Neoparamoeba perurans and fish were sampled at 36 days postinoculation (pi.). AGD-affected gill tissue was dissected from N. perurans infected fish, and a DNA microarray was used to compare global gene expression against tissues from AGD-naive fish. To determine whether the changes in gene expression were restricted to AGD-lesions, lesion tissue from AGD-affected fish was also compared with non-lesion gill tissue dissected from the same gill arch. Samples were assessed using a DNA microarray.
Keywords: comparative gene expression, parasite-induced lesion, Neoparamoeba perurans, amoebic gill disease

overall_design: mRNA from lesion and non-lesion gill tissue was amplified and labeled. Six biological and two technical replicates were utilised to hybridise to 12 arrays using amplified RNA from AGD-affected lesion gill tissue with AGD-naive fish as a control. Four biological and two technical replicates were utilised to hybridise to 8 arrays using amplified RNA from AGD-affected lesion gill tissue with non-lesion tissue from the same gill arch as a control. The assignment of microarrays to treatment groups for hybridization was randomised by using a random number generator.

first: Neil

last: Young

first: Glenn

middle: A

last: Cooper

first: Barbara

last: Nowak

last: Morrison


title: Convergence of TCR and IL-7 pathways leads to FOXO3a phosphorylation and drives the survival of C. Memory CD4+ T cells

status: Public on Jan 09 2007

city: Cleveland

first: Peter

last: Wilkinson

state: Ohio

address: 2103 Cornell Road

department: Pathology / Systems Biology & Bioinformatics

country: USA

institute: Case Western Reserve University

summary:
The molecular events that are involved in the establishment and the maintenance of CD4+ Central Memory (TCM) and Effector Memory (TEM) T cells are poorly understood. Using global gene expression profiling, single cell proteomics, and functional assays, we show that the survival of TCM CD4+ T cells involves the activation and phosphorylation of STAT5a and FOXO3a. STAT5a phosphorylation induces the transcriptional up-regulation of anti-apoptotic genes specifically in TCM. The phosphorylation of FOXO3a at S315, following TCR engagement, prevents the transcription of pro-apoptotic gene like FasL and BIM. Experiments aimed at blocking FOXO3a phosphorylation confirmed the role of FOXO3a in protecting TCM from apoptosis. Our results define the underlying molecular mechanisms responsible for the longevity and persistence of CD4+ TCM.
Keywords: comparative gene profile, cell-type comparison, central memory to Effector memory

overall_design: Isolation of CD4+T cell sub-populations. Peripheral blood mononuclear cells (PBMCs) from healthy adult individuals were isolated by Ficoll-HyPaque (Pharmacia) density gradient. We first enriched for CD4+ T cells using negative immunomagnetic beads selection (Automacs, Myltenii), cells were then labeled with anti-CD4-APCcy7, anti-CD45RA-ECD, anti-CD27-FITC and anti-CCR7-PEcy7 and sorted into Naive cells described as CD4+, CD45RA+, CD27+ and CCR7+, Central Memory cells (TCM) described as CD4+, CD45RA-, CD27+ and CCR7+ cells and Effector Memory cells (TEM) described as CD4+, CD45RA-, CD27- and CCR7- cells. Sorting was performed using a BDAria (BD Pharmingen).  Purity of the TCM and TEM sub-populations was ranging from 96 to 99%. All procedures were done at 4Â°C to avoid any changes in cell phenotype or gene expression. Sample RNA was extracted using an RNA extraction kit (Quiagen), then amplified using the MessageAmp RNA kit (Ambion) as per the manufacturer's instructions. The amplified RNA (aRNA) was then verified for quality and quantity using the Agilent Bioanalyser and measuring the OD. Universal human RNA (Stratagene) was also prepared in the same way.  Sample probes were prepared by direct labelling with 3 µg of the aRNA Cy-5 (R values) fluorescent dye while the universal RNA probes were prepared by direct labelling of universal aRNA with Cy-3 (G values). All patient samples were hybridized against amplified universal RNA at 37 ºC for 18h on a custom human Immune array. Detailed information on the labeling and hybridization procedures can be obtained at URL:  http://www.microarrays.ca.

first: C

last: Riou

first: B

last: Yassine-Diab

first: J

last: Van grevenynghe

first: R

last: Somogyi

first: L

last: Greller

first: P

first: Y

last: Shi

first: D

last: Kelvin

first: M

last: Cameron

last: Balderas

middle: P

last: Sekaly

first: E

middle: K

last: Haddad


title: Cis-transcriptional variation in maize inbred lines B73 and Mo17 leads to additive expression

status: Public on Jun 21 2007

city: Saint Paul

first: Nathan

middle: M

last: Springer

state: MN

address: 1445 Gortner Ave

department: Plant Biology

institute: University of Minnesota

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

overall_design: Refer to individual Series


title: Genetic alteration in recurrent melanoma

status: Public on Jul 22 2008

city: Doha

first: Francesco

middle: Maria

last: Marincola

address: Al Nasr Tower, AL Corniche Street, PO Box 26999

country: Qatar

institute: Sidra Medical and Research Center

summary:
It is generally accepted that human cancers derive from a mutated single cell. However, the genetic steps characterizing various stages of progression remain unclear. Studying a unique case of metastatic melanoma, we observed that cell lines derived from metachronous metastases arising over a decade retained a central core of genetic stability in spite of divergent phenotypes. In the present study we expanded our previous observations comparing these autologous cell lines of clonal derivation with heterologous ones and correlated array Comparative Genomic Hybridization (aCGH) with gene expression profiling to determine their relative contribution to the dynamics of disease progression. aCGH and gene expression profiling were performed on autologous cell lines and heterologous melanoma cell lines originated from other patients. A striking correlation existed between total extent of genetic imbalances, global transcriptional patterns and cellular phenotypes; they did not follow a strict temporal progression but stemmed independently at various time points from a central core of genetic stability best explained according to the cancer stem cell hypothesis; although their contribution was intertwined, genomic imbalances detectable by aCGH contributed only 25% of the transcriptional traits determining autologous tumors distinctiveness. Our study provides important insights about the dynamics of cancer progression and supports the development of targeted anti-cancer therapies against stable genetic factors determining the individuality of each patient’s disease that are maintained throughout the end stage of disease.
Keywords: genetic modification design

overall_design: PBMC from a female donor were Ficoll gradient separated and used throughout the study as reference.. Validation of array CGH accuracy was done by obtaining six additional PBMC from female donors and six PBMC from male donors to confirm stability of gene representation in autosomes and sex-determined imbalances within the X and Y chromosome regions. Five melanoma cell lines were generated from distinct cutaneous melanoma metastases that progressively appeared in patient 888. Other melanoma cell lines were generated from cutaneous melanoma metastases from other patients. The melanoma cell line A375 was purchased from the American Type Culture Collection. For each cell line arrayCGh has been performed.


title: Prediction of graft-versus-host disease in humans by donor gene expression profiling

status: Public on Mar 27 2007

summary:
Graft-versus-host disease (GVHD) results from recognition of host antigens by donor T cells following allogeneic hematopoietic cell transplantion (AHCT). Notably, histoincompatibility between donor and recipient is necessary but not sufficient to elicit GVHD. Therefore, we tested the hypothesis that some donors may be “stronger alloresponders” than others, and consequently more likely to elicit GVHD. To this end, we analyzed the gene expression profile of CD4 and CD8 T cells from 50 AHCT donors. We found that pre-AHCT gene expression profiling segregates donors whose recipient suffered from GVHD or not. The “dangerous donor” trait (GVHD+ recipient) is under polygenic control and is shaped by the activity of genes that regulate diverse cell functions including TGF-β signaling and cell proliferation. We also performed gene expression profiling in T cells harvested from 40 AHCT recipients on day 365 post-AHCT. The donor gene profile defined on day 0 showed exceedingly strong correlation with that of recipient CD4 and CD8 T cells harvested one year post-AHCT. These data suggest that donor gene profiles linked with GVHD were largely imprinted at the hematopoietic stem cell level. The ability to discriminate strong and weak alloresponders using gene expression profiling could pave the way to personalized transplantation medicine.
Keywords: comparative gene profile, cell type comparaison, GVHD+, GVHD-

overall_design: Peripheral blood was obtained from 50 AHCT donors pre-transplant (referred to as day 0) and from 40 recipients on day 365. Donors and recipients were HLA-identical siblings. Recipients were regarded as negative for acute GVHD (aGVHD) when they lived at least 100 days without presenting GVHD. Recipients were considered negative for chronic GVHD (cGVHD) when they remained cGVHD-free for 365 days post-AHCT. CD4 and CD8 T-cell subsets were purified with microbeads. Total RNA was purified, amplified, reverse transcribed and hybridized on microarrays developed by The Microarray Centre of The Toronto University Health Network (http://www.microarrays.ca/). RNA from donor and recipient T cells was hybridized on the human H19K array (19,008 ESTs), and donor T-cell RNA was also hybridized on the ImmunArray (3,411 ESTs from immune related genes).

first: Chantal

last: Baron

first: Roland

first: Larry

middle: D

first: Vincent

last: Rineau

first: Carolyn

middle: R

last: Cho

first: Mark

middle: J

first: David

first: Pierre

last: Chagnon

first: Denis-Claude

last: Roy

first: Lambert

last: Busque

first: Rafick-Pierre


title: p53 status and BaP or BPDE gene expression response

status: Public on Nov 13 2007

city: Sutton

first: Ian

last: Giddings

state: Surrey

address: 15 Cotswold Road

department: Section of Molecular Carcinogenesis

country: United Kingdom

institute: Institute of Cancer Research

summary:
Human colon carcinoma cells (HCT116) differing in p53 status were exposed to benzo(a)pyrene (BaP) (2.5 and 5 uM for up to 48 h) or anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE)(0.5 and 1 uM for up to 24 h), and their gene expression responses compared by cDNA microarray technology.
Keywords: BaP or BPDE exposure

overall_design: 1 samples  - cells exposed to BaP or BPDE dissolved in DMSO were compared to cells exposed to DMSO alone


title: Toxicogenomic responses in rainbow trout hepatocytes exposed to model chemicals and a synthetic mixture

status: Public on Dec 05 2006

first: Ben

middle: F

last: Koop

state: BC

department: Biology

country: Canada

summary:
Comparison of gene expression of exposed versus non-exposed Oncorhynchus mykiss hepatocytes to four model chemicals and a synthetic mixture.  Hepatocytes were exposed for 24 hours to a single chemical and a synthetic mixture of 10 nM 17 alpha-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 μM paraquat and 0.75 μM 4-nitroquinoline-1-oxide (NQO).
Keywords: Exposed vs. control

platform_organism:
Osmerus mordax
Oncorhynchus mykiss
Salmo salar
Coregonus clupeaformis
Oncorhynchus tshawytscha

overall_design: Four biological replicates for both exposed and non-exposed Oncorhynchus mykiss hepatocytes with corresponding dye flips.

first: Eivind

last: Finne

first: Glenn

last: Cooper

first: Alan

last: Wardroper

first: Ketil

last: Hylland

first: Knut-Erik

last: Tollefsen


title: Common genome-wide patterns of transcript accumulation underlying the wing polyphenism and polymorphism in the pea aphid

status: Public on Jun 05 2007

city: Princeton

first: Gregory

middle: K.

last: Davis

state: NJ

address: 106A Guyot Hall, Washington Road

department: Ecology and Evolutionary Biology

institute: Princeton University

summary:
The pea aphid, Acyrthosiphon pisum, exhibits several environmentally cued polyphenisms, in which discrete, alternative phenotypes are produced. At low density parthenogenetic females produce unwinged female progeny, but at high density females produce progeny that develop with wings. These alternative phenotypes represent a solution to the competing demands of dispersal and reproduction. Males also develop as either winged or unwinged, but these alternatives are determined by a genetic polymorphism. Winged and unwinged males are morphologically less distinct from each other than winged and unwinged females, possibly because males experience fewer trade-offs between dispersal and reproduction. To assess whether shared physiological differences mirror the shared morphological differences that characterize the wing polyphenism and polymorphism, we used a cDNA microarray representing an estimated 10% of the coding genome (1734 genes) to examine differential transcript accumulation between winged and unwinged females and males. We identified several transcripts that differentially accumulate between winged and unwinged morphs in both sexes, the majority of which are involved in energy production. Unexpectedly, the extent of differential transcript accumulation between winged and unwinged morphs was greater for adult males than for adult females. Together, these results suggest not only that similar physiological differences underlie the polyphenism and polymorphism, but that male morphs, like females, are subject to trade-offs between reproduction and dispersal that are reflected in levels of transcript accumulation and possibly genome-wide patterns of gene regulation. These data also provide a baseline for future studies of the molecular and physiological basis of life history trade-offs.
Keywords: Transcript levels were compared between winged and unwinged male and female pea aphids, for both nymphs and adults.

platform_organism:
Buchnera aphidicola
Escherichia coli
Acyrthosiphon pisum

overall_design: Labeled cDNA made from RNA isolated from winged and unwinged male and female pea aphids (nymphs and adults) was hybridized to a spotted cDNA microarray (1734 aphid genes) against a common referent. Three biological replicates of each aphid type (winged state, sex, stage) were performed.

first: Jennifer

last: Brisson

middle: L

last: Stern


title: Expression profiling of zmet2-m1 mutants relative to wild-type

status: Public on Dec 01 2007

first: Nathan

last: Springer

summary:
The contribution of epigenetic alterations to natural variation for gene transcription levels remains unclear.  In this study, we investigated the functional targets of the maize chromomethylase ZMET2 in multiple inbred lines to determine whether epigenetic changes conditioned by this chromomethylase are conserved or variable within the species.  Gene expression microarrays were hybridized with RNA samples from the inbred lines B73 and Mo17, and from near-isogenic derivatives containing the loss-of-function allele zmet2-m1.  A set of 126 genes that displayed statistically significant differential expression in zmet2 mutants relative to wild-type plants in at least one of the two genetic backgrounds were identified.  Analysis of the transcript levels in both wild-type and mutant individuals revealed that only 10% of these genes were affected in zmet2 mutants in both B73 and Mo17 genetic backgrounds.  Over 80% of the genes with expression patterns affected by zmet2 mutations display variation for gene expression between wild-type B73 and Mo17 plants.  Further analysis was performed for seven genes that were transcriptionally silent in wild-type B73, but expressed in B73 zmet2-m1, wild-type Mo17 and Mo17 zmet2-m1 lines.  Mapping experiments confirmed that the expression differences in wild-type B73 relative to Mo17 inbreds for these genes were caused by cis-acting regulatory variation.  Methylation-sensitive PCR and bisulphite sequencing demonstrated that for five of these genes the CpNpG methylation in the wild-type B73 genetic background was substantially decreased in the B73 zmet2-m1 mutant and in wild-type Mo17.  A survey of eight maize inbreds reveals that each of these five genes exhibit transcriptionally silent and methylated states in some inbred lines and unmethylated, expressed states in other inbreds, providing evidence for natural variation in epigenetic states for some maize genes.
Keywords: mutant versus wild-type comparison in two inbred genotypes

overall_design: The zmet2-m1 mutant allele was backcrossed into two inbred backgrounds, B73 and Mo17.  RNA was isolated from 6 biological replicates of B73 wild-type plants, 6 biological replicates of B73 zmet2-m1 mutant plants; 3 biological replicates of Mo17 wild-type plants and 3 biological replicates of Mo17 zmet2-m1 mutant plants.

first: Irina

last: Makarevitch

first: Robert

last: Stupar


title: Cross-Platform Array Comparative Genomic Hybridization (array CGH) Meta-Analysis

status: Public on Aug 29 2006

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

country: Netherlands

institute: Vrije Universiteit Medical Center

type: Genome variation profiling by genome tiling array

summary:
A series of studies have been published that evaluate the chromosomal copy number changes of different tumor classes using array Comparative Genomic Hybridization (array CGH), however the chromosomal aberrations that distinguish the different tumor classes have not been fully characterized. Therefore, we performed a meta-analysis of different array CGH data sets in an attempt to classify samples tested across different platforms. As opposed to RNA expression a common reference is used in dual channel CGH arrays: normal human DNA, theoretically facilitating cross-platform analysis. To this aim, cell line and primary cancer data sets from three
different dual channel array CGH platforms obtained by four different institutes were integrated. The cell line data were used to develop preprocessing methods which performed noise reduction
and transformed samples into a common format. The transformed array CGH profiles allowed perfect clustering by cell line, but importantly not by platform or institute. The same preprocessing
procedures used for the cell line data were applied to data from 373 primary tumors profiled by array CGH, including controls. Results indicated that there is no apparent feature related to the
institute or platform and that array CGH allows for unambiguous cross-platform meta-analysis. Major clusters with common tissue origin were identified. Interestingly, tumors of hematopoietic
and mesenchymal origins cluster separately from tumors of epithelial origin. Therefore it can be concluded that chromosomal aberrations of tumors from hematopoietic and mesenchymal origin versus tumors of epithelial origin are distinct, and these differences can be picked up by metaanalysis of array CGH data. This suggests the possibility of prospectively using combined analysis of diverse copy number datasets for cancer subtype classification.
Keywords: comparative genomic hybridization, meta-analysis, cancer

overall_design: 20 cell lines and 140 tumor samples.

first: Kees

last: Jong

first: Elena

last: Marchiori

first: Aad

last: van der Vaart

first: Chin

last: Suet-Feung

first: Beatriz

last: Carvalho

first: Marianne

last: Tijssen

first: Paul

last: Eijk

last: van den IJssel

first: Heike

last: Grabsch

first: Philip

last: Quirke

first: Joost

last: Oudejans

first: Gerrit

last: Meijer

first: Carlos

last: Caldas

first: Bauke

last: Ylstra


title: Cell line stimulated with recombinant cytokines interleukin-1b and interferon gamma.

status: Public on Jun 16 2007

city: Aberdeen

first: Sam

last: Martin

state: Scotland

address: Tillydrone Avenue

department: Zoology

institute: University of Aberdeen

summary:
Interleukin 1b and interferon g are two cytokines that are central to the inflammatory response.  They both have a broad range of activity, each directing the immune response to be towards either the innate response (IL-1b and IFN-g) or the cellular response (IFN-g), however invivo there is a lot of subtle interactions.  Microarray analysis has been used to characterize the transcriptional response to recombinant rainbow trout IL-1b and IFN-g in a rainbow trout macrophage cell line RTS-11.  The use of the cell line removes the host response and the interference of other cellular factors that occur when performing whole fish experiments, or even primary cultures.  RNA was extracted from stimulated or control cells following 6h incubation, this was used to hybridize to a 16K v1 salmonid cDNA array constructed by GRASP consortium.
Keywords: Dual label cDNA microarray

platform_organism:
Oncorhynchus mykiss
Salmo salar

overall_design:
Rainbow trout cell line RTS-II was stimulated with either recombinant trout interleukin 1 beta or recombinant trout interferon gamma, or not stimulated.
Total RNA was extracted from cells 6 h following stimulation.
Three flasks were used for each cytokine and total RNA was isolated.
6 slides were used for interleukin 1 beta stimulation which was the 3 samples and dye swaps.
6 slides were used for interferon gamma which was the 3 samples and dye swaps.

first: Samuel

first: Jun

last: Zou

first: Dominic

last: Houlihan

first: Chris

last: Secombes


title: Analysis of gene expression in rice mature leaf and cultured cell suspension using Rice SuperSAGE Array1

status: Public on Jul 28 2006

city: Ueda

first: Hideo

last: Matsumura

state: Nagano

address: Tokita 3-15-1

department: Gene Research Center

country: Japan

institute: Shinshu University

summary:
We developed a novel platform for genome-wide gene expression analysis in any eukaryotic organism, that we coin SuperSAGE Array. The SuperSAGE Array is a microarray onto which oligonucleotides of 26 nucleotides corresponding to SuperSAGE tag sequences are directly synthesized.  A SuperSAGE Array combines the advantages of the highly quantitative SuperSAGE expression analysis with the high-throughput microarray technology.  Highly reproducible gene expression profiling by the SuperSAGE Array was demonstrated for a thousand of genes (tags) in rice.
Keywords: Tissues, SuperSAGE, 26base oligonucleotide

overall_design: SuperSAGE Array was produced by on-chip synthesis of 26-base long oligonucleotides exactly corresponding to the tag sequences.


title: Identification of genes regulated by RORg in mouse thymus

status: Public on Apr 01 2006

city: RTP

first: NIEHS

last: Microarray Core

state: NC

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

summary:
Microarray analysis was performed in order to identify changes in gene expression in thymocytes isolated from RORg-/- mice in comparison to those of wild type mice.
Keywords: Genetic Modification

overall_design: Microarray analyses were carried out by the NIEHS Microarray Group (NMG). Gene expression analysis was conducted using Agilent Mouse arrays (Agilent Technologies, Palo Alto, CA) representing about 20,000 genes. For each condition, equal amounts of total RNA from thymocytes of three individual wild type or RORg-/- mice were pooled and amplified. Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol.  Starting with 1 mg of total RNA, Cy3- or Cy5-labeled cRNA was synthesized according to manufacturer’s protocol.  For each comparison, 750 ng of each Cy3- and Cy5-labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol.  Hybridizations were performed for 17 h in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol.  Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner.  Data was obtained with the Agilent Feature Extraction software (v7.1) using defaults for all parameters.

first: HongSoon

last: Kang

first: JuYoun

last: Beak

first: Yong-Sik

last: Kim

first: Robert

last: Petrovich

first: Jennifer

middle: B

last: Collins

first: Sherry

last: Grissom

first: Anton

last: Jetten


title: Cis-transcriptional variation in maize inbred lines B73 and Mo17 leads to additive expression - Seedling data

first: Nathan

last: Springer

summary:
Microarray analysis of gene expression patterns in immature ear, seedling, and embryo tissues from the maize inbred lines B73 and Mo17 identified numerous genes with variable expression. Some genes had detectable expression in only one of the two inbreds; most of these genes were detected in the genomic DNA of both inbreds, indicating that the expression differences are likely caused by differential regulation rather than by differences in gene content. Gene expression was also monitored in the reciprocal F1 hybrids B73xMo17 and Mo17xB73. The reciprocal F1 hybrid lines did not display parental effects on gene expression levels. Approximately 80% of the differentially expressed genes displayed additive expression patterns in the hybrids relative to the inbred parents. The approximately 20% of genes that display nonadditive expression patterns tend to be expressed at levels within the parental range, with minimal evidence for novel expression levels greater than the high parent or less than the low parent. Analysis of allele-specific expression patterns in the hybrid suggested that intraspecific variation in gene expression levels is largely attributable to cis-regulatory variation in maize. Collectively, our data suggest that allelic cis-regulatory variation between B73 and Mo17 dictates maintenance of inbred allelic expression levels in the F1 hybrid, resulting in additive expression patterns.
Keywords: genotype comparison

overall_design: Affymetrix expression profiling was used to study gene expression in aerial tissue from 11-day seedlings of maize.  Three biological replicates were performed for four different genotypes; B73, Mo17, B73xMo17 and Mo17xB73.

first: Stupar

last: Robert


title: Evidence of off-target effects associated with long dsRNAs in Drosophila cell-based assays

status: Public on Aug 18 2006

city: Boston

first: Meghana

last: Kulkarni

state: MA

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary:
To evaluate the specificity of long dsRNAs used in high-throughput RNAi screens performed at the Drosophila RNAi Screening Center (DRSC), we performed a global analysis of their activity in 30 genome-wide screens completed at our facility. Surprisingly, our analysis predicts that dsRNAs containing ≥19 nucleotide perfect matches identified in silico to unintended targets may contribute to a significant false positive error rate arising from off-target effects. We confirmed experimentally that such sequences in dsRNAs lead to false positives and to the efficient knockdown of a cross-hybridizing transcript, raising a cautionary note when interpreting results based on the use of a single dsRNA per gene. Although a full appreciation of all causes of false positive errors remains to be determined, we suggest simple guidelines to help ensure high quality information from RNAi high-throughput screens
Keywords: Specificity of long dsRNAs, Drosophila melanogaster, SL2 cell line, custom cDNA arrays, off-target effects, Drosophila RNAi Screening Center (DRSC)

overall_design:
Custom cDNA microarrays were used to compare the transcriptional signature elicited by the PP2A-B’ dsRNA, DRSC16337 (D1), to those generated by 3 additional PP2A-B’ dsRNAs (C1, C2 and C3) in Drosophila melanogaster SL2 culture cells. Three independent experiments were set up for each dsRNA experiment. To avoid dye bias during labeling of cDNA we performed dye swaps. Cell culture and dsRNA experiments: dsRNA was generated and used essentially as previously described (Clemens, J. C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U S A 97, 6499-503 (2000). Briefly, gene-specific primers were designed to include T7 promoter sequences used in vitro transcription of the PCR products (T7 MEGAscript Kit; Ambion) to generate both RNA strands in one reaction. The dsRNAs were purified with RNeasy columns (Qiagen) and quality controlled by agarose gel electrophoresis.
Drosophila Schneider SL2 cells were cultured in Schneider’s medium (GibcoBRL) supplemented with 10% fetal bovine serum (JRH Bioscience) and Penicillin-Streptomycin (GibcoBRL) at 25 degree C, unless noted otherwise. 4x106 SL2 cells were seeded in each well of a 6-well cell culture dish (Falcon) in 1ml of serum-free media and treated with 15 µg of dsRNA at room temperature for 45 min. After 45 min, 2 ml of serum-supplemented Schneider’s medium was added and cells were further incubated for 72 hr. For expression profiling experiments three independent treatments were setup for each dsRNA. Total RNA was isolated by Trizol according to the manufacturer’s specifications (GibcoBRL), purified with RNeasy columns (Qiagen) and quality was assessed using the Agilent Bioanalyzer according to manufacturer’s specifications (Agilent). Custom cDNA-based Microarray: DNA probes to a set of approximately 3,000 strategically selected genes were generated by PCR. These probes were obtained from the PCR amplicons used to generate the dsRNA library 16. The 3,000 genes represented on the array include genes for all the Kinases (350) and Phosphatases (161) encoded in the Drosophila genome, known/canonical components and targets of the major signaling pathways such as Wg/Wnt, Hh, TGF-β, JAK/STAT, NFκB, Notch, RTK, JNK, and INR pathways; (http://genome.med.yale.edu/Lifecycle). Approximately 1,000 of the 3,000 genes were randomly selected to facilitate normalization of signal intensity across the array. Only PCR products showing a clear and strong bands were recovered using Montage PCR plates (Millipore). The DNA concentration was determined using PicoGreen reagent (Molecular Probes). The final concentration of DNA averaged 400 ng/microliter. Samples were transferred to 384 well plates (Genetix: x6004) containing DMSO to give a final concentration of 200 ng/microliter in 50% DMSO. The DNA probes were directly spotted onto Corning UltraGAPS coated glass slides using a Genetix QArray robot in the Biopolymer Facility at Harvard Medical School. Spots were arrayed in a 14 x 14 arrangement using 24 (4 x 6) Genetix 150 microm diameter tip solid microarray pins with a center-to-center spacing of 315 microm. A number of controls were prepared and printed on the glass slides with the probe DNAs. These controls include Negative controls (water, 50% DMSO, sonicated salmon sperm DNA, human Cot1, mouse Cot1, GFP, CFP, YFP, LacZ, and Luciferase) to assess the degree of nonspecific hybridization, and Spiked controls (Lucidea Universal Scorecard, Amersham Biosciences).

first: Matthew

last: Booker

first: Serena

last: Silver

first: Adam

last: Friedman

first: Pengyu

last: Hong

first: Norbert

last: Perrimon

first: Bernard

last: Mathey-Prevot


title: Microarray-based CGH in human lymphoma cell lines.

status: Public on Feb 05 2007

city: Pamplona

first: Jose Angel

middle: Martinez

last: Climent

state: Navarra

address: PIO XII

country: Spain

institute: CIMA/Universidad De Navarra

summary:
This series represents the data set of Array CGH from the paper (submit for publication): “Homozygous deletions localize novel tumor suppressor genes in B-cell lymphomas. Mestre et all. ” and it's web supplement.
The molecular nature of many secondary events in the pathogenesis of B-cell non Hodgkin lymphoma (B-NHL) remains unknown. We used high-resolution CGH to BAC microarrays to characterize the genomes of 48 B-cell non-Hodgkin lymphoma (B-NHL) cell lines of different origins.
Array CGH identified, on average, 20 genomic alterations per cell line, including regional gains and losses as well as previously uncharacterized aberrations.  Different genomic patterns were observed among the B-NHL subtypes. To search for possible oncogenic target genes, gene expression profiling was performed in the cell lines models.
Integrative genomic and gene expression analyses have identified amplified oncogenes in B-cell non-Hodgkin lymphoma (B-NHL), but the capability of such technologies to localize tumor suppressor genes within homozygous deletions remains unexplored. Array-based comparative genomic hybridization (CGH) and gene expression microarray analysis of 48 cell lines derived from patients with different B-NHLs delineated twenty homozygous deletions at seven
chromosome areas, all of which contained tumor suppressor gene targets. Further investigation revealed that only a fraction of primary biopsies presented inactivation of these genes by point mutation or intragenic deletion, but instead some of them were frequently silenced by epigenetic mechanisms. Our microarray strategy has identified novel candidate suppressors inactivated by genetic and epigenetic mechanisms that substantially vary among the B-NHL subtypes.
Keywords: Array CGH, B-cell non-Hodgkin lymphoma, genomic amplification, homozygous deletion

overall_design:
Genome-wide analysis of DNA-copy number changes of the 48 cell lines was performed using array CGH on a microchip with 2.400 BAC and P1 clones in triplicate. The array provides an average resolution of 1.4 Mb across the genome. Production and validation of the array, hybridization methods and analytical procedures have been described elsewhere in detail.( Snijder AM, Nowak N, Segraves R, et al. Assembly of microarrays for genome-wide measurement of DNA copy number. Nat Genet. 2001;29:263-264.)  Briefly, 0.2 µg of test (tumor) and reference genomic DNAs were labeled by random priming using Cy3 and Cy5, respectively.  After 48 hour hybridization, slides were washed and mounted with DAPI.  The images of the arrays were captured using a CCD camera, and the “UCSF SPOT” software was used to analyze the images.  A second program “SPROC” was used to associate clones with each spot and a mapping information file that allows the data to be plotted relative to the position of the BACs on the draft human genome sequence (http://genome.cse.ucsc.edu; July 2003 freeze).
A formal data filtering procedure was then performed, and a SPROC output file consisting of averaged ratios of the triplicate spots for each clone, standard deviations of the replicates and plotting positions for each clone on the array, was obtained.  For visualization of genomic data, the TreeView program 1.60 (Stanford, CA) was used.
Mean log2 ratios (tumoral DNA vs. control DNA) were plotted and the resultant graphs were converted to the log2 domain. The observed log2 ratios were excluded from further analysis if there were fewer than two replicate spots (out of 3) or if the standard deviation of the replicates was above 0.2. The clones that were present in fewer than 50% of the samples were also removed from the dataset. The following mean log2 ratios (tumoral DNA vs. control DNA) were used to classify genomic aberrations: genomic gain (between 0.39 and 0.95), genomic amplification (greater than 0.95) or genomic loss (lower than -0.5 for heterozygosity, and lower than –1.4 for homozygous deletion). Statistically significant correlations between the genomic imbalances and the B-NHL subgroups were analyzed using Fisher’s exact test.  For delineation of common regions of genomic imbalance in the lymphoma genomes, the position of the BAC/PACs on the draft human genome sequence according to the May 2004 freeze was used (http://genome.cse.ucsc.edu; May 2004 freeze).


title: Gender associated differential gene expression in Schistosoma mansoni adult worms

status: Public on Aug 24 2006

city: Campos dos Goytacazes

first: Thiago

middle: Motta

last: Venancio

state: RJ

address: Av Alberto Lamego 2000

country: Brazil

institute: UENF

summary:
The complex molecular systems involved in the process of sex-differentiation and fertility in Schistosoma mansoni have not yet been completely described.  Using a 4608-element cDNA microarray, we have now determined 90 and 139 genes with significantly (q-value ≤ 0.06) higher expression levels in adult males and females, respectively. Eight out of eleven selected transcripts had their differential expression levels validated by Real-Time RT-PCR. One of these transcripts was extended by RT-PCR and was shown to span  the intronic region between exons 9 to 11 of the S. mansoni CA150 gene, a transcriptional cofactor known in humans to interact with both RNA polymerase II and the spliceosome complex. The longer transcript probably represents a novel isoform of S. mansoni CA150.  Additionally, we obtained full-length sequences for 3 other isoforms of the SmCA150 gene, coding for proteins of different lengths and domain compositions. Semi-quantitative RT-PCR showed different expression ratios among these isoforms between male and female. Due to the role of CA150 in RNA transcription and processing, we hypothesize that these differential expression events may be important in the generation and maintenance of the different phenotypes between male and female.
Keywords: comparative gender gene expression

overall_design: Three separate samples from males and three from female were analyzed. For each sample, materials from 3 perfusions performed at different dates were pooled, and male and female parasites were separated manually. Two biological replicate sample were prepared with the same procedure, each using material from 3 independent perfusions. One technical replicate from one of the samples was obtained for each gender.

first: Ricardo

last: DeMarco

first: Katia

middle: C

last: Oliveira

first: Sergio

last: Verjovski-Almeida


title: Cohesin and cohesion establishment factors distribution on S.Cerevisiae chromosome VI during the cell cycle

status: Public on Sep 02 2006

city: Montpellier

first: Armelle

last: Lengronne

address: 141 rue de la Cardonille

department: Institut de Genetique Humaine

country: France

institute: CNRS

type: Genome binding/occupancy profiling by genome tiling array

summary:
Two identical sister copies of eukaryotic chromosomes are synthesised during S-phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from their synthesis onwards by the chromosomal cohesin complex. It is thought that replication fork progression is coupled to establishment of sister chromatid cohesion, thus facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4 and Ctf18 are found close to, and Ctf4 travels along chromosomes with replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S-phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment, and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved.
Keywords: ChIP on chip

overall_design:
Number of hybridizations performed: 33 ChIP analysis

Samples used, extract preparation and labelling:

• The origin of each biological sample: Saccharomyces cerevisiae (W303 background)
• Manipulation of biological samples and protocols used:
Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VI were performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature).
• Technical protocols for preparing the hybridization extract
The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization.

Hybridization procedures and parameters:

•Hybridization, blocking and washing were carried out as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3’biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix).

Measurement data and specifications:

•Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All processed data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-value≤0.025). Secondly, reliability of binding ratio was judged by change p-value (p-value≤0.0025). Thirdly, clusters consisting of at least 900bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors.

Array Design:
•General array design: in situ synthesized arrays by Affymetrix
Availability of arrays: commercially available from Affymetrix
Location and ID of each spot on arrays: available from Affymetrix on request
•Probe type: oligonucleotide
The arrays used in this study can be purchased from Affymetrix:
Chromosome VI S.cerevisiae: rikDACFC6, P/N# 510636

first: John

last: McIntyre

first: Yuki

last: Katou

first: Yutaka

last: Kanoh

first: Karl-Peter

last: Hopfner

first: Katsuhiko

last: Shirahige

first: Frank

last: Uhlmann


title: Atlantic Salmon Amoebic Gill Disease Study

status: Public on Dec 20 2005

first: Ben

last: Koop

summary:
Comparison of gene expression of infected versus non infected Salmo salar gill tissue over time.  The time course includes 0 hr, 44 hr, 114 hr and 189 hr post infection
Keywords: Infected vs control

overall_design: Three biological replicates for both infected and non infected Salmo salar were measured for each time point.

first: Richard

middle: N

last: Morrison

first: Glenn

last: Cooper

first: Matthew

last: Rise

first: Andrew

last: Bridle

first: Mark

last: Adams

first: Barbara

last: Nowak


title: Gene expression in human vs chimpanzees

status: Public on Jun 08 2005

city: Stanford

last: Stanford Microarray Database

state: CA

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

summary:
Despite the close evolutionary relationship and striking genetic similarity between humans and chimpanzees, there are remarkable differences in anatomy, behavior, and disease susceptibility in the two species. One step towards understanding the biological basis of these phenotypic differences is to characterize quantitative differences in levels of expression of genes in humans and chimpanzees. To contribute to such analysis, we compared gene expression patterns in lymphoblastoid cell lines between nine unrelated humans and ten unrelated chimpanzees by using human cDNA microarrays. Hybridizations to arrays containing 43,233 features produced high quality data for 22,879 cDNA clones, representing 20,266 Unigenes. We observed statistically significant differences in transcript levels for 32% of these genes (P < 0.05, Student t test), with about 200 cDNAs showing differences of more than 2-fold (lower bounds of 95% confidence interval). Among these are genes involved in cell surface glycosylation and responses to toxins and viruses. Examination of functional annotations for the differentially expressed genes revealed lower expression of cell cycle and energy pathways genes, and higher expression of chemokines, 26S proteasome and cell motility genes in chimpanzee samples. These genes and pathways could underlie some of the phenotypic differences between humans and chimpanzees.
Keywords: other

sample_organism:
Pan troglodytes
Homo sapiens

first: J

last: Zhang


title: Differences found in expression of cell surface & ECM components during development of the rainbow trout ovary & testis.

status: Public on Jun 29 2007

summary: A comparison of "maturing" and "prespawn" ovarian and testicular transciptomes was performed to determine the genes that are involved in regulating gametic and accessory cell function during maturation and development of the rainbow trout gonad.  To identify some fo the genes involved in these processee, total RNA was compared between three-year-old normal vs two-year-old normal (maturing) and three-year-old normal vs two-year-old precocious (prespawn) gonadal tissue.

overall_design: Three biological replicates for both "maturing" and "prespawn" rainbow trout ovary and testis with corresponding dye flips.

first: Kris

last: von Schalburg

first: William

middle: S

last: Davidson

first: Jong

last: Leong

first: Ryosuke

last: Yazawa


title: Human Heterochromatin Proteins form Large Domains Containing KRAB-ZNF Genes

status: Public on Oct 16 2006

first: Bas

last: van Steensel

address: Plesmanlaan 121

department: division of Molecular Biology

institute: Netherlands Cancer Institute

type: Expression profiling by genome tiling array

summary:
Heterochromatin is important for gene regulation and chromosome structure, but the genes that are occupied by heterochromatin proteins in the mammalian genome are largely unknown. We have adapted the DamID method to systematically identify target genes of the heterochromatin proteins HP1 and SUV39H1 in human and mouse cells. Unexpectedly, we found that HP1beta (CBX1) and SUV39H1 bind to genes encoding KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. These genes constitute one of the largest gene families and are organized in clusters in the human genome. Preference of CBX1 for this gene family was observed in both human and mouse cells. High-resolution mapping on human Chromosome 19 revealed that CBX1 coats large domains 0.1 - 4Mb in size, which coincide with the position of KRAB-ZNF gene clusters. These domains show an intricate CBX1 binding pattern: while CBX1 is globally elevated throughout the domains, it is absent from the promoters and binds more strongly to the 3’ ends of KRAB-ZNF genes. KRAB-ZNF domains contain large numbers of LINE elements, which may contribute to CBX1 recruitment. These results uncover a surprising link between heterochromatin and a large family of regulatory genes in mammals. We suggest a role for heterochromatin in the evolution of the KRAB-ZNF gene family.
Keywords: DamID, chromatin profiling, DNA microarray, expression profiling.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
In this study we mapped the genomic binding sites of heterochromatin proteins CBX1, CBX3, CBX5 and SUV39H1 in different cell types using the DamID method.
DamID involves the low level expression of a fusion protein consisting of DNA adenine methyltransferase (Dam) and a chromatin protein of interest. This fusion protein is targeted to the native binding sites of the chromatin protein, where Dam methylates adenines in the surrounding DNA. The methylated DNA fragments were isolated and amplified by selective PCR, labeled with a fluorescent dye and hybridized to microarrays.
In this study we used 3 different array platforms for detection of amplified methylated DNA fragments. All experiments were done with samples obtained from independent experiments and include dye swaps.
In addition, we made an mRNA expression profile of MCF7 cells (4 arrays)

first: Maartje

last: Vogel

first: Lars

last: Guelen

first: Elzo

last: de Wit

first: Daniel

last: Peric Hupkes

first: Martin

last: Loden

first: Wendy

last: Talhout

first: Marike

last: Feenstra

last: Abbas

first: Anne-Kathrin

last: Classen


title: MogR regulon in response to temperature and growth condition

status: Public on Feb 11 2006

first: Aimee

last: Shen

address: Armenise Building, rm 313, 200 Longwood Avenue

department: Microbiology and Molecular Genetics

institute: Harvard University

summary:
Microarray analyses comparing the transcriptional profile of wild-type EGDe relative to ∆mogR (L. monocytogenes) during growth at 37˚C and RT in BHI broth and during intracellular growth inside J774 host cells at 37˚C. These analyses revealed that MogR primarily functions to repress flagellar motility genes during growth at 37˚C.
Keywords: EGDe vs ∆mogR, Temperature, Growth condition

overall_design: Oligonucleotide spotted arrays obtained from the Pathogen Functional Genomic Resource Center at TIGR were used for competitive hybridization studies comparing the transcriptional profile of wild-type EGDe to ∆mogR (L. monocytogenes). A minimum of two technical replicates with associated dye swaps was performed for each growth condition. 14 samples were analyzed, comprising 9 biological replicates; dye swaps were performed for 6 of the biological replicates.

first: Darren

middle: E

last: Higgins


title: Altered gene expression profiles in testis developmental exposed to endocrine disruptors

status: Public on Jun 01 2010

city: Madrid

first: Luis

middle: A.

last: Lopez-Fernandez

address: Dr. Esquerdo 46

department: Pharmacy

institute: Hospital General Universitario Gregorio Marañon

summary: Environmental reproductive health focuses on the effect of exposure to contaminants considered as endocrine disruptors. Developmental testis is considered as target of these compounds affecting testicular functions in adults and suspected implications in tumor etiology. Comparative analysis of gene expression in mouse testis exposed to five disruptors, three different dosages and three accumulative developmental stages shown defined signature profiles of gene deregulation for MEHP (monoethyl phthalate) and zearalenone (a phytoestrogen) and different to 17β-estradiol exposure. The effects are even detected in postpuberal male offspring from premating exposed mothers. Oxidative stress response, protein ubiquitination and oxidative phosphorylation are the most representative pathways affected.

overall_design:
Animal Exposure

	CD-1 mice were in vivo exposed to several doses of EDs following a defined protocol: mothers were exposed two weeks before mating (exposure A); the same exposure and dose were maintained during pregnancy (exposure B) and four weeks after birth (exposure C). In all exposures, male offspring were sacrificed to obtain the testes.
	The compounds were administrated in drinking water to a final concentration of:

        	E2(mg/l)	BPA(mg/l)	Zea(mg/l)	MEHP(mg/l)	Lindane(mg/l)
Dose (1)	   0.02	  0.5          	4	       27.83	             50
Dose (2)	   0.04	  50	              12	     139.17	           100
Dose (3)	   0.16	  200	              20	     278.34	           200

	Ethanol was used as vehicle for E2, BPA and Zea and DMSO was for MEHP and Lindane. Control testes were obtained from animals exposed to solvents of EDs: Ethanol and DMSO.
	Animal care and sacrifice were made following regulations from the CSIC Bioethics Committee.

RNA preparation

	Total RNA from testes of exposed animals and controls was extracted using TRIzol reagent (Invitrogen), according to the manufacturer’s standard protocol. RNA samples were spectrophotometrically quantified using NanoDrop (NanoDrop Technologies). To minimize the effect of inter-individual expression variability we made pools of RNA from at least 3 individual RNA samples from each experimental condition (same quantity of total RNA per sample). Pools of RNA were checked for quality performing Experion analysis (Bio-Rad Laboratories).
	Total RNA from testes of unexposed four weeks old animals was also extracted with the purpose of making pools to assess the variability at the expression level of the mouse strain used (Ctr).

Microarray Processing

	Starting with total RNA from pools described above, full length cDNAs were synthesized from oligo(dT) primers bearing a T7 promoter. These cDNAs were used as template for in vitro transcription by T7-RNA-Polymerase to amplify the mRNAs; following the Amino Allyl MessageAmp™ aRNA Kit (Ambion, Inc.) protocol. The amplified mRNAs (aRNA) were labeled either with Cy3 or Cy5 dyes, using Amersham CyDye Post-Labeling Reactive Dyes (Amersham Biosciences). The incorporated dye was spectrophotometrically quantified using NanoDrop (NanoDrop Technologies). The same dye signal quantities of labeled experimental and control aRNAs were mixed and hybridized to Mouse Oligoset v3 (OPERON) arrays from Genomics facility-University of Cincinnati (USA). The slides contained 31,769 spotted probes (70 mer oligonucleotides) corresponding to 24,878 expressed or predicted mouse genes. Hybridization and washes of microarrays were performed according to Genomics facility-University of Cincinnati instructions.
	Dye-Swap replicates were performed: one slide hybridized with experimental and control targets labeled with Cy3 and Cy5, respectively; and the second slide with the same type of targets but inversely labeled. Target refers to labeled aRNA while probe refers to the oligonucleotides spotted on the microarray platform.
	Imaging and data generation were carried out using a GenePix 4000B (Axon Instruments) and associated software from Axon Instruments, Inc. The microarray slides were scanned with dual lasers with wavelength frequencies to excite Cy3 and Cy5. Images were captured in TIFF files. Information extraction for a given spot was based on the median value for the signal pixels minus the median value for the background pixels to produce a gene set data file for all the DNA spots. The Cy3 and Cy5 fluorescence signal intensities were normalized.

first: Pedro

middle: P

last: Lopez-Casas

middle: A

first: Maria

last: Paz

first: Jesus

last: del Mazo