Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Differential expression of MEN2 versus VHL human Pheochromocytomas

ABSTRACT: This study utilized oligonucleotide microarrays to examine gene expression profiles in pheochromocytomas from 23 patients; 11 VHL tumors and 12 MEN2 tumors. RNA was extracted and linearly amplified once, then dye-labeled and co-hybridized to a microarray with over 34,000 oligos representing more than 25,000 unique human genes. In validation of our strategy, many genes known to have elevated expression in MEN2 tumors (e.g., CALM1, CHGB, CABP1and NPY) and several genes known to be specific to VHL (e.g., AQP1) were independently identified by this approach. In addition to known genes, our method has identified several hundred of previously uncharacterized genes that are enriched in the MEN2 or VHL tumors. A 34k expression arrays were generated by printing Oligo nucleotides from the Qiagen HV3 set onto epoxy coated slides (Corning, Inc). Total RNA from pheochrmocytoma tumors or normal adrenals was amplified using an Ambion kit (Cat#1753), labeled with Cy5 and cy3 dyes respectively and hybridized overnight to the oligo chip in a MAUI chamber.

ORGANISM(S): Homo sapiens

SUBMITTER: Christopher Pan

PROVIDER: E-GEOD-12642 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification.

Brown Jamie L JL Snir Mirit M Noushmehr Houtan H Kirby Martha M Hong Sung-Kook SK Elkahloun Abdel G AG Feldman Benjamin B

Proceedings of the National Academy of Sciences of the United States of America 20080821 34

A major goal for developmental biologists is to define the behaviors and molecular contents of differentiating cells. We have devised a strategy for isolating cells from diverse embryonic regions and stages in the zebrafish, using computer-guided laser photoconversion of injected Kaede protein and flow cytometry. This strategy enabled us to perform a genome-wide transcriptome comparison of germ layer precursor cells. Mesendoderm and ectoderm precursors cells isolated by this method differentiate ...[more]

PMID: 18719100

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Project description:We have developed a method for rapidly identifying early, regionally-expressed molecules: FACS-Assisted Microdissection of Photolabeled cells (FAM-P). For our first FAM-P project, we injected embryos with purified Kaede protein, a stony coral protein that fluoresces at 514 nm (green) prior to- and 582 nm (red) after photoconversion at 405 nm. We used the scanning laser of a confocal microscope to photolabel late blastula-stage embryos along 4-6 tiers of marginal cells, comprising the mesoderm and endoderm germ layer precursors. This procedure left the neurectoderm precursor cells marked by green fluorescence and the mesendoderm precursor cells marked by red fluorescence. Embryonic cells were dissociated and subjected to FACS, separating red mesendoderm precursors from green neurectoderm precursors. RNA was extracted and linearly amplified once, then dye-labeled and co-hybridized to a microarray with over 30,000 oligos representing more than 20,000 unique zebrafish genes. In validation of our strategy, many genes known to have elevated expression in the late blastula margin (e.g., squint, fgf8, lim1 and gata5) and several genes known to be specific to the early neurectoderm (e.g., sox31 and otx2) were independently identified by this approach. In addition to known genes, our method has identified dozens of previously uncharacterized genes that are enriched in the pre-mesendoderm or pre-neurectoderm. Keywords: 40% epiboly-stage embryos, injected with purified Kaede protein, differentially photolabeled, disaggregated and FACS sorted 34k expression arrays were generated by printing Oligo nucleotides from three different sets (MWG, Compugen and Operon) onto epoxy slides. Total RNA from Kaede protein-injected and FAM-P treated zebrafish embryos was amplified using an Ambion kit (Cat#1753), labeled with Cy dyes and hybridized overnight to the oligo chip in a MAUI chamber. The experiment comparing mesendoderm was performed four times (twice with WIK/AB hybrid strain embryos, once with EK strain embryos and once with AB strain embryos) each time including a dye swap, for a total of eight (four forward and four dye-swap) hybridizations. Three control cRNA probes were also generated from AB-strain embryos, namely: (1) whole 40% epiboly stage embryos, (2) FACS-sorted green cells from dissociated 40% epiboly embryos that were Kaede-injected but NOT photolabeled and (3) FACS-sorted red cells from dissociated 40% epiboly embryos that were Kaede-injected AND photolabeled. These probes were each generated three times, each time including a dye swap and co-hybridized as follows: (1) whole embryo cRNA vs green cell cRNA (three forward and three dye-swap hybridiizations) and (2) green cell cRNA vs. red cell cRNA (three forward and three dye-swap hybridizations).

Project description:Gene expression profiling of tissues derived from different genetic backgrounds can provide insight to potential phenotype-related changes in gene expression. We have compared wildtype C57BL/6 (melan-Ink4a) melanocyte cell gene expression to that of, âbrownâ Tyrp1b/ b (melan-Ink4a-b) and âchocolateâ Rab38 cht/cht (melan-cht) melanocyte derived lines. We observed alterations in gene expression consistent with a physiological response of melan-Ink4a-b cells to endoplasmic reticulum protein-folding impairment. Additionally, a set of loci was identified that are expressed in a similar manner to known melanocyte genes. Importantly, the collection of 254 genes is enriched for expression in the multiple melanocyte lines relative to control cell populations. This set contains Tyr, Tyrp1, Dct, Si, Melna, Slc24a5, Slc45a2, Slc24a4, Mitf, and Sox10, in addition to genes not previously attributed to pigmentation and genes with unknown function. Further evaluation of one gene in this cohort, Gpnmb, demonstrated an expression pattern similar to Dct, Si and Mitf. This expression is vastly reduced in MitfMi mutants embryos, indicating that it is dependent on Mitf for in vivo expression. Consistent with this notion, a highly-conserved MITF binding site resides directly upstream of human GPNMB, and deletion of this sequence dramatically reduces in vitro enhancer activity. Keywords: NIH embryo fibroblast cell line, melanocyte cell line, Ink4a, Tyrp1b, Rab38 A 32k expression arrays were generated by printing Oligo nucleotides from Operon Mouse oligo set (MV3) onto epoxy slides. Total RNA from immortalized melanocyte cell lines derived from C57BL/6J Ink4a-/- (melan-Ink4a-1), C57BL/6J Ink4a-/-; Tyrp1b/b (melan-Ink4a-b) and 2 separate C57BL/6J Ink4a-/-; RaB38cht/cht lines (melan-cht4), (melan-cht5) and NIH3T3 were labeled with Cy5/3 and co-hybridized with a total RNA universal reference (Stratagene, Inc) labeled with Cy3/5. Each comparison was done in Triplicate including one dye swap.

Project description:Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIAs) are analogues of the products of PI3K that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of 5 active PIAs and the PI3K inhibitor LY294002 (LY) was performed in non-small cell lung cancer (NSCLC) cells using high-density oligonucleotide arrays. Gene ontology analysis revealed genes involved in apoptosis, wounding response, and angiogenesis were upregulated by PIAs, while genes involved in DNA replication, repair and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into 3 groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3 and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB) and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity. H157 cells were plated at 2x10^6 in T-75 flasks in RPMI medium 1640 containing 10% FBS and incubated for 24h. The media was then changed to RPMI medium 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM PIA6 dissolved in DMSO for 0h, 2h, 6h or 12h, and an equal volume of DMSO was added to control samples. For the PIA comparison, 10 microM PIAs (5, 6, 7, 23, 24, 25) or 10 microM LY294002 (LY) were incubated with the cells for 6h. In the PIA comparison experiment, the same concentration of PIA7 (an inert analog consisting of only the lipid side chain) was used as a control. Following incubation, the alterations in cellular morphology were photographed, and cells from 6-well plates were harvested for immunoblot analysis. Total RNA was extracted from cells treated in T-75 flasks using TRIzol reagent (Invitrogen) and chloroform, and purified according to the RNeasy midiprep spin kit protocol (Qiagen). Oligonucleotide microarray was performed with dye-swap. Microarray chips were generated from the 34,580 longmer probe set Human Genome Oligo Set Version 3.0 (Qiagen). Protocols for cDNA labeling, hybridization, and scanning are available through the National Human Genome Research Institute microarray core.

Dataset Information

Differential expression of MEN2 versus VHL human Pheochromocytomas

Publications

Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification.

Similar Datasets

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