Comparison of expression profile between human MM-CM-
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Caroline Lupien
PROVIDER: E-GEOD-3118 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Comparison between the gene expression profile of human Müller cells and two spontaneous Müller cell lines.
Lupien Caroline B CB Bolduc Carl C Landreville Solange S Salesse Christian C
Investigative ophthalmology & visual science 20071101 11
<h4>Purpose</h4>Müller cells are the principal glial cells in the retina. They play important functions in this tissue. Alterations in Müller cell behavior were observed in the retinal tissue of patients with proliferative diabetic retinopathy. The purpose of this study was to determine the gene expression profile of normal human Müller cells (NHMCs) and to compare it with that of two spontaneously generated human Müller cell lines (HMCLs) from type 1 and type 2 diabetic donors by using serial a ...[more]
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Project description:This study characterizes the expressed transcripts of 15 intact tissues in mice by using the serial analysis of gene expression (SAGE) strategy which indicates the relative level of expression for each transcript matched to the tag. Keywords: double-strand cDNA , serial analysis of gene expression (SAGE) , ditags Total RNA was isolated from tissues by using the RNA extraction kit (TRIzol Reagent, Invitrogen Canada Inc., Burlington, ON). Approximately 5 µg of mRNA was extracted with Oligotex mRNA Mini Kit (Qiagen Inc., Mississauga, ON). The SAGE method was performed as previously described (Velculescu et al., 1995; St-Amand et al., 2001). In brief, double-strand cDNA was synthesized from the mRNA using a biotinylated (T)18 primer and cDNA synthesis kit (Invitrogen Canada Inc.). The cDNA libraries were digested with the restriction enzyme NlaIII (New England Biolabs Inc., Pickering, ON). The 3'-terminal cDNA fragments were captured using streptavidin-coated magnetic beads (Dynal, Biotech LLC, Brown Deer, WI). After ligation of 2 annealed linker pairs, the cDNA fragments were digested with BsmFI (New England Biolabs Inc.). The blunting kit from Takara Bio Inc. (Otsu, Japan) was used for the blunting and ligation of the two tag populations. The resulting ligation products were amplified by PCR and digested with NlaIII. The band containing the ditags was extracted from the 12% polyacrylamid gel. Using T4 ligase (Invitrogen Canada Inc.), the ditags were self-ligated to form concatemers that were cloned into SphI site of pUC19. White colonies were screened by PCR and agarose gel to select long inserts for automated sequencing (Applied Biosystems 3730, Foster City, CA). The sequence and occurrence of each tag were analyzed by the SAGEana program, which is a new version of SAGEparser.pl (Dinel et al., 2005).
2007-04-03 | E-GEOD-5915 | biostudies-arrayexpress
Project description:This study describes the application of the LongSAGE methodology to study the gene expression profile in Leishmania infantum chagasi. A tag library was created using the LongSAGE method and consisted of 14,208 tags of 17 bases. Of these, 8,427 (59.3%) were distinct. BLAST research of the 1,645 most abundant tags showed that 12.8% of them identified the coding sequences of genes, while 82% (1,349/1,645) identified one or more genomic sequences that did not correspond with open reading frames. Only 5.2% (84/1,645) of the tags were not aligned to any position in the L. infantum genome. The UTR size of Leishmania and the lack of CATG sites in some transcripts were decisive for the generation of tags in these regions. LongSAGE revealed the gene expression profile in promastigotes of L. i. chagasi. Additional analysis will allow a better understanding of the expression profile and discovering the key genes in this life cycle. The I-SAGETM Long kit (Invitrogen) was used to construct the L. i. chagasi library according to the manufacturer's recommendations. The following were included among the main steps: polyadenylates mRNAs were captured by oligo (dT) linked to magnetic beads and used for cDNA synthesis. The cDNA was digested with 60 U of NlaIII (anchoring enzyme) and the 3' ends of the cDNAs were isolated using the beads. The resulting cDNA 3' was divided into two equal portions and connected to two LongSAGE adapters, A and B. The long tags were released by the enzyme MmeI and linked to form ~130 bp ditags. Dilutions of 1:40 of the product were amplified in 27 PCR cycles (300 reactions in total). The precipitated PCR products were separated on 12% polyacrylamide gel and the 130 bp bands were cut out and the precipitated DNA was digested with NlaIII. The digestion products were separated on 12% polyacrylamide gel and ~34 bp ditags were cut out and linked to form concatemers. The concatemers were separated on 6% polyacrylamide gel and the fractions of 250-500 and 500-800pb were isolated and cloned into pZErO-1 vector digested with SphI. Vectors with concatemers were cloned in E. coli DH10b by electroporation and the isolated recombinant vectors were used as template for sequencing in the ABI Prism 3100 (Applied Biosystems).
2011-08-09 | E-GEOD-29369 | biostudies-arrayexpress
Project description:We identified 3418 genes transcribed at a level of at least two copies each. We identified many transcripts involved in protein translation, cell maintenance and metabolism, as expected for vegetative cells. The most highly expressed cell signaling genes include ubiquitin, smlA, and nucleotide exchange factors RasGEF F and Ras GEF G. Additionally, we identified many genes previously reported to be expressed only during later stages of development including dutA, actin8, thioredoxin3, culmination specific protein 45D, discoidin II and yelA. We isolated total RNA from Dictyostelium amoeba growing in HL5 media with shaking and at a density of 2.4 X 10(7) cells/ml using Ambion RNAqueous isolation kit. We prepared the SAGE library using an Invitrogen kit and being careful to reduce the amount of ditags in the concatemer ligation reaction to 25% of the manufacturer's suggested mass. Concatemers were sequenced at the Molecular Research Core Facility on the campus of Idaho State University using an Applied Biosystems 3100 Genetic Analyzer. We identified SAGE tags by comparison to the Dictyostelium genome sequence available on the dictybase website.
2008-12-31 | E-GEOD-12095 | biostudies-arrayexpress
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.
2009-05-20 | E-GEOD-16139 | biostudies-arrayexpress
Project description:The cochlear nucleus is the first central pathway involved in the processing of peripheral auditory activity. It is heterogeneous in neuronal populations and physiologic responses and is organized in three major subdivisions: the anterior ventral cochlear nucleus (AVCN), the posterior ventral cochlear nucleus (PVCN) and the dorsal cochlear nucleus (DCN). Although each region demonstrates multiple cell types and functions, there are predominant populations of neurons in each region that underlie the principal role each subdivision plays in auditory processing. Little is known of the underlying genetic contribution to these properties. This study sought to identify genes expressed in the subdivisions of the cochlear nucleus that may account for the anatomical and physiological characteristics of each subdivision. These data provide a genetic basis for understanding normal auditory processing in the cochlear nucleus and a template for investigating changes that may occur with hearing loss, the generation and percept of tinnitus, and central processing disorders. Keywords: normal, comparative Brown Norway rats (n=40, female, 45days) were anesthetized and decapitated. Brains were rapidly removed and the subdivisions of the cochlear nucleus (AVCN, PVCN and DCN) dissected on dry ice. Total RNA was extracted and tested for concentration and purity by spectrophotometry and integrity by gel electrophoresis. SAGE was performed using the NlaIII enzyme and Invitrogen SAGE kit. Concatemers were commercially sequenced and imported into eSAGE (Margulies and Innis, 2000) for tag extraction and frequency.
2005-12-31 | E-GEOD-3628 | biostudies-arrayexpress
Project description: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 the GermLine, Alpha) and demonstrated its involvement in two independent developmental processes: formation of the primordial follicle and coordinate expression of zona pellucida genes. Taking advantage of Figla null mouse lines, we have used a combined approach of microarray and Serial Analysis of Gene Expression (SAGE) to identify potential downstream targets genes. Using high stringent cutoffs, we find that FIGLA functions as 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. Keywords: Serial Analysis of Gene Expression The libraries were constructed using Sau3AI as the anchoring enzyme as described in the SAGE protocol (Cheval et al., 2002). Total RNA (~50 µg) was obtained from newborn normal and Figla null ovaries with an RNeasy mini kit (Qiagen, Valencia, CA, USA). The presence of MSY2 and the absence of ZP2 transcripts as detected by one-step qRT-PCR (Qiagen, Valencia, CA, USA) confirmed the identity of Figla null ovaries. Poly(A)+ RNA was isolated with oligo d(T)-conjugated magnetic beads (Dynal-Invitrogen, Calsbad, CA, USA). SAGE libraries are constructed as per the protocol described by Virlon B and group (Virlon B et al, 1999).Briefly, cDNA was synthesized from 5 μg of poly(A) RNAs with the cDNA synthesis system kit. Double stranded cDNA was synthesized using the Invitrogen SuperscriptII kit. The double-strand cDNA was digested with Sau3AI, and the 3′ end was isolated through binding to 1 mg of paramagnetic streptavidin beads (Dynal, Invitrogen, Calsbad, CA, USA), The streptavidin-bound cDNA was divided into two fractions. Each fraction was ligated to 10 pmol of either linker 1 or linker 2, then digested with BsmFI. The released cDNA tags were blunt-ended for 10 min at 42°C in a 50-μl reaction volume containing 20 mM Tris HCl (pH 7.5), 10 mM MgCl2, 25 mM NaCl, 10 mM DTT, 400 μM dNTP, and 10 units of T7 DNA polymerase (Pharmacia Biotech). The two fractions then were ligated to each other by using T4 DNA ligase. PCR (95°C, 30 sec; 58°C, 30 sec; 70°C, 45 sec: 26–28 cycles) was carried out on 1% of ligation reaction in a 100-μl reaction volume containing 20 mM Tris HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 4 mM DTT, 100 μM dNTP, 50 pmol of primers 1 and 2, and 5 units of Taq polymerase. The products from 10–12 reactions were pooled. The 110-bp DNA fragment was purified by agarose gel electrophoresis and submitted to preparative PCR (120–150 reactions performed as described above, except that the amount of primers was reduced to 25 pmol and the number of cycles was 12). The PCR sample was digested with Sau3AI, and ditags were purified and concatenated as described (Velculescu et al, 1995). Concatenated ditags were cloned into Bluescript pKS (Stratagene, La Jolla, CA, USA) using the BamHI site and blue-white screening, prior to sequence, identified colonies with inserts. Plating, picking, DNA preparation and sequencing were performed at the the NIH Intramural Sequencing Center (NISC). Low quality sequence data were eliminated by Phred analysis.
2007-09-07 | E-GEOD-5802 | biostudies-arrayexpress
Project description:The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment by SAGE analysis. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unabl e to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g., an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modeling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors. SAGE analysis of C. neoformans cells isolated from macrophages The murine macrophage-like cell line J774A.1 was maintained at 37M-BM-0C in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS), 1% nonessential amino acids, 100 M-BM-5g mL-1 penicillin-streptomycin, and 4 mM L-glutamine (Invitrogen). Cryptococcus cells were opsonized with monoclonal antibody 18B7 against capsule (1 M-BM-5g mL-1), and macrophages were treated with recombinant mouse gamma interferon (IFN-gamma) (50 U mL-1) and lipopolysaccharide (LPS) (0.3 M-BM-5g mL-1) prior to co-incubation at a multiplicity of infection (MOI) of 1:1. Macrophages were inoculated with H99 cells and washed after 1 h of inoculation to remove unattached, extracellular fungal cells. After 6 h of incubation, sterile, ice-cold distilled H2O was applied to each well to lyse the macrophages, and the fungal cells (4 x 107) were harvested by centrifugation. H99 control cells were prepared by growth under the same condition but without macrophages, and 108 cell s were harvested. SAGE library construction, sequencing and analysis SAGE library construction, sequencing and analysis were as previously described (Hu et al., 2007; Hu et al., 2008; Steen et al., 2002; Steen et al., 2003). Briefly, RNA was isolated from lyophilized cells by vortexing with glass beads (3.0 mm, acid-washed and RNase-free) for 15 min in 15 mL of TRIZOL extraction buffer (Invitrogen, Carlsbad, CA, USA). The mixture was incubated for 15 min at room temperature and total RNA was isolated according to the manufacturer's instructions. Total RNA was used directly for SAGE library construction as described by Velculescu et al. (1995) using the I-Long SAGE kit (Invitrogen). The tagging enzyme for cDNA digestion was NlaIII and 29 PCR cycles were performed to amplify the ditags during library construction. Colonies were screened by PCR (M13F and M13R primers) to assess the average clone insert size and the percentage of recombinants. Clones from the libraries were sequenced by BigDye primer cycle sequencing on an ABI PRISM 3700 DNA analy zer. Sequence chromatograms were processed using PHRED (Ewing et al., 1998), and vector sequence was detected using Cross_match (Gordon et al., 1998). Fourteen-base-pair tags were extracted from the vector-clipped sequence, and an overall quality score for each tag was derived based on the cumulative PHRED score. Duplicate ditags and linker sequences were removed as described previously (Steen et al., 2002). Only tags with a predicted accuracy of 99% were used, and statistical differences between tag abundance in different libraries were determined as described (Audic and Claverie, 1997). The libraries yielded 23,904 tags from cells grown in media without macrophages for 6hr and 38,5444 tags from cells grown in media with macrophages for 6hr. An overview of the abundance classes for both SAGE libraries is presented in Table S1 in the supplementary material, with both the number of different tag sequences and the total number of tags present in each class for the cells from th e 6h infection and the control. All libraries were normalized to 23,904 to allow direct comparisons, and the tags that appeared less than once in any given library were removed. The EST database available for strain H99 at the University of Oklahoma's Advanced Center for Genome Technology http://www.genome.ou.edu/cneo.html) was used for the preliminary assignment of tags to genes. When an EST sequence could not be identified for a particular tag, the genomic sequence for H99 at the Duke University Center for Genome Technology (http://cgt.genetics.duke.edu/data/index.html) and the Broad Institute (http://cneo.genetics.duke.edu/) was used to identify contigs with unambiguous tag assignments. Note that a limitation of the SAGE approach is that some transcripts are not detected because of low abundance and/or the absence of an NlaIII site for transcript processing. The control (fungal cells alone) and cryptococcal-macrophage interaction SAGE datasets were deposited to the NCBI under accession number GSM986206 and GSM986207 respectively.
2013-08-15 | E-GEOD-41066 | biostudies-arrayexpress
Project description:In this study, we sequenced 691,390 SAGE tags from four libraries. Cervical L-SAGE libraries N1, N2, C1, and C2 were sequenced to 165,624, 181,224, 173,534, and 171,008 tags, respectively. Duplicate ditags were eliminated from analysis resulting in 136,276, 139,656, 154,828 and 136,386 useful tags respectively and a total of 24 058 unique tags. 15,438 of the unique tags mapped to annotated UniGene identifiers. We characterized the transcriptome of normal cervical tissue and evaluated the highly expressed genes in terms of tissue specificity, conserved expression among the normal libraries and their altered expression in CIN III lesions. Keywords: Cervical Epithelium, Long SAGE Four Long SAGE libraries were created from cervical epithelium biopsies. Two were CIN III and two were normal cervical tissue.
2008-02-18 | E-GEOD-6252 | biostudies-arrayexpress
Project description:To identify new candidate genes for neurocristopathies, a Long-SAGE library has been made from human neural crest cells (hNCC) derived from a normal embryo at Carnegie stage 13. Data analysis indicates the expression of a number of genes in multiple GO functional classes that were anticipated based on animal studies and/or human disease. An average linkage hierarchical clustering was performed using SAGE data from hNCC and 14 other normal tissues and cell lines. This and other analyses indicate a high degree of molecular identity between hNCC and the two human embryonic stem cells (hES) lines included, compared to the other tissue or cell types included, including more tissue-restricted stem cells, such as mesenchymal stem cells, or hNCC derivatives such as Schwann cells. Keywords: Cell type comparison Total RNA was extracted from a confluent 10 cm dish of hNCC (line N5) from a female human embryo at 28-32 dpf (Carnegie stage 13). Half the RNA was used to construct a SAGE bank with long tags using NlaIII and MmeI. Data was then compared to currently publicly available SAGE data from fourteen banks made from normal human tissues for hierarchical clustering. Third-party GEO accession numbers for human SAGE data: GSM41360, hES3; GSM41362, hES4; GSM48250, sciatic nerve; GSM48251, Schwann cells (in vitro); GSM31931, brain substantia nigra; GSM824, muscle; GSM785, liver; GSM708, kidney; GSM676, brain white matter; GSM761, brain cerebellum; GSM762, lung. Other third-party data: Cancer Genome Anatomy Project http://cgap.nci.nih.gov/SAGE/SAGELibraryFinder: SAGE_Prostate_normal_B_2 (prostate) Umbilical cord- and bone marrow-derived mesenchymal stem cell data had been downloaded from currently broken links http://bit.fmrp.usp.br/uc-msc_tags/ and http://bit.fmrp.usp.br/msc_tags/ as referenced in Panepucci et al. (2004) Stem Cells 22:1263-1278; DOI: 10.1634/stemcells.2004-0024 (UC-MSC) and Silva et al. (2003) Stem Cells 21:661-669.
2008-08-07 | E-GEOD-8368 | biostudies-arrayexpress
Project description:This study characterizes the expressed transcripts of 15 intact tissues in mice by using the serial analysis of gene expression (SAGE) strategy which indicates the relative level of expression for each transcript matched to the tag. Keywords: double-strand cDNA , serial analysis of gene expression (SAGE) , ditags
2007-04-03 | GSE5915 | GEO