Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Rosetta (Merck) Expression Validated Gene Hybridizations

ABSTRACT: Rosetta (Merck) Expression Validated Gene Hybridizations

ORGANISM(S): Homo sapiens

SUBMITTER: Eric Schadt

PROVIDER: E-GEOD-918 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A comprehensive transcript index of the human genome generated using microarrays and computational approaches.

Schadt Eric E EE Edwards Stephen W SW GuhaThakurta Debraj D Holder Dan D Ying Lisa L Svetnik Vladimir V Leonardson Amy A Hart Kyle W KW Russell Archie A Li Guoya G Cavet Guy G Castle John J McDonagh Paul P Kan Zhengyan Z Chen Ronghua R Kasarskis Andrew A Margarint Mihai M Caceres Ramon M RM Johnson Jason M JM Armour Christopher D CD Garrett-Engele Philip W PW Tsinoremas Nicholas F NF Shoemaker Daniel D DD

Genome biology 20040923 10

<h4>Background</h4>Computational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays c ...[more]

PMID: 15461792

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Project description:Samples were purchased from Clontech as mRNA. Hybridization material was generated through a random-priming amplification procedure (RP-AMP) using primers with a random sequence at the 3' end and a fixed motif at the 5' end. shDNP256 (first strand synthesis): 5'- TAG ATG CTG TTG NNN NNN NNN -3' shT7N9 (second strand synthesis): 5'- ACT ATA GGG AGA NNN NNN NNN -3' 1.5 g of mRNA was reverse-transcribed with Superscript II and the DP256 random primer for 20 minutes at 42 C (10 mM DTT, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 5 U/ l Superscript II). The RNA was degraded with the addition of 20 l volume of 0.5N sodium hydroxide and 0.25 M EDTA for 20 minutes at 65 C. The single stranded cDNA was purified using a commercial kit (Qiagen Qiaquick). The resulting product of single stranded cDNA was placed in its entirety in a second strand reaction. Second strand synthesis reactions utilized shT7N9 random primer and the Klenow fragment of DNA polymerase utilizing standard reaction conditions (37 C for 60 minutes, 0.2 mM DTT, 2.1 mM Tris-HCl pH 7.9, 2.1 mM MgCl2, 10.7 mM NaCl, 1.07 mM dNTPs, 0.1U/ l Klenow), followed by another Qiaquick purification. Multiple polymerase chain reactions were run using 0.15 g of double stranded DNA and standard reaction conditions. Amplification was achieved using 10 cycles of PCR with the DP256 and T7 primers (20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.01 mM dNTPs, 1.5 mM MgCl2, 0.01 U/ l Taq Polymerase) DP256: 5'- GTT CGA GAC CTC TAG ATG CTG TTG -3' T7: 5'- AA TTA ATA CGA CTC ACT ATA GGG AGA -3' followed by Qiaquick purification. Further amplification was achieved using in vitro transcription reactions with X0.5 g dsDNA and T7 RNA Polymerase (7.5 mM DTT, 40 mM Tris-HCl pH 7.5, 14.25 mM MgCl2, 10 mM NaCl, 2 mM Spermidine, 125 U/ml RNAguard, 2.5 mM dNTPs, 15 U/ml IPPase, 25kU/ml T7 Polymerase) for 16 hours at 42 C. The cRNA was purified (RNeasy) and reverse transcribed using Superscript and random 9-mers and amino-allyl dUTP (42 C for 20 minutes, 10 mM DTT, 50mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 0.5 mM aa-dUTP, 5 U/ l Superscript II). The cDNA for each channel was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 1M sodium bicarbonate, pH 9.0, followed by quenching with 4.0 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were purified with a QIAquick PCR purification kit and the percentage dye incorporation and total cDNA yield was determined spectrophotometrically. Cy5 and Cy3 labelled cDNAs were combined and added to 2 mls 30% formamide with Herring Sperm DNA for hybridization to the microarray. Further details can be found in Castle et al., Genome Biol 2003;4(10):R66, http://genomebiology.com/2003/4/10/R66 , Roberts CJ et al., Science 2000, 287:873-880, and Schadt, Edwards, et al., unpublished. Normalization of single channel data is described in Ying, et al., "Identification of Chromosomal Regions Containing Transcribed Sequences Using Microarrays and Computational Methods" , 2003 Proceedings of the American Statistical Association, Biopharmaceutical Section [CD-ROM], Alexandria, VA: American Statistical Association, 2003. Briefly, an invariant set of probes was selected based on their intensity ranks. Then, a LOESS regression model was fit for Cy5 vs. Cy3 intensities for all probes in the invariant set. Finally, for all probes on the array the Cy5 intensities were normalized to the corresponding Cy3 intensities using prediction values from the regression model. Note that the normalized intensity values given here were used for all analyses reported in the accompanying paper. The ratio values reported here were not used for the analysis described in the manuscript and are not calculated from the normalized intensity values given below.

Project description:Adult mouse subventricular zone (SVZ) neural progenitor cells (NPCs) retain the capacity to generate multiple lineages in vitro and in vivo. Thus far, the mechanisms involved in the regulation of these cells have not been well elucidated. We have carried out RNA profiling of adult SVZ cell cultures undergoing differentiation, to identify pathways that regulate progenitor cell proliferation and to define a set of transcripts that can be used as molecular tools in the drug discovery process. We carried out a stepwise stratification of the results to identify transcripts specifically enriched in NPCs and validated some of these using comparative literature analysis, quantitative PCR and immunological techniques. The results show a set of transcription factors, secreted molecules, and plasma membrane markers which are differentially regulated during differentiation. Pathway analysis highlights alterations in IGF, Wnt and TGFbeta signalling cascades. Further characterization of these components could provide greater insight into the mechanisms involved in the regulation of neurogenesis in the adult brain. Mouse subventricular zone neural stem cells cultures were prepared from C57Bl6/J mice (Johansson et al.,1999) and cultured in suspension in DFEM/F12 1:1 supplemented with PSF, B27 supplement and EGF/ FGF-2 at 20ng/ml. RNA was isolated from either expanding neurospheres in the presence of FGF2 and EGF (Reynolds and Weiss, 1992; Gritti et al., 1999), or from cultures induced to differentiate upon growth factor withdrawal or serum exposure, either after 24 hours (in suspension, to avoid changes induced by substrate attachment) or 5 days (as adherent cultures in poly-lysine and laminin-coated plates). Experiments were conducted in triplicate from independent batches of SVZ preparations. As a control for potential effects of media changes, a set of identical cultures were included where cells were grown for a further 24 hours in fresh media containing FGF2 and EGF (20 ng/ml each, normal growth media, NGM). Fluorescently labelled aRNA from individual samples were competitively hybridised against a pool of labelled aRNA from undifferentiated reference (starting) material on custom Agilent microarrays representing ~22,500 mouse transcripts. Technical, fluor-reversed hybridisations were performed for every sample.

Project description:COPD is a common and disabling lung disease for which very few therapeutic options are currently available. We reasoned that global gene expression profiling of COPD lungs could reveal previously unidentified disease pathways for potential therapeutic interventions. Forty-eight human lung samples were obtained from lungs or lobes resected for small peripheral lung lesions (5 non-smokers, 21 GOLD 0, 9 GOLD 1, 10 GOLD 2 and 3 GOLD 3 patients). mRNA from the specimens was profiled using Agilent’s Functional ID v2.0 array which contains 23,720 sequences. Quantitative morphometric analysis of the specimens revealed that the samples contained a variable proportion of airways, blood vessels and parenchyma. Incorporating these data into a model relating gene expression to % predicted forced expiratory flow between 25 and 75% of forced expiratory volume (FEF 25-75 % P) revealed a signature gene set of 203 transcripts. Genes involved in extracellular matrix synthesis/degradation, oxidative stress and cell proliferation were among the up-regulated genes whereas genes which participate in nicotine metabolism, elastic fiber homeostasis and anti-inflammatory response were down-regulated. Immunohistochemistry confirmed expression of urokinase (PLAU), urokinanse receptor (PLAUR) and thrombospondin (THBS1) by alveolar macrophages and small airway epithelial cells. Genes in this pathway have been shown to be involved in transforming growth factor beta-1 (TGF?1) and matrix metalloproteinase (MMP) activation and are subject to inhibition by serpin E2. Interestingly, both TGF?1 and serpin E2 have been identified as candidate genes in COPD genetic linkage and association studies. The results thus provide a possible link between these two powerful approaches to identify potential therapeutic targets. (255 words) Forty-eight human lung samples were obtained from lungs or lobes resected for small peripheral lung lesions (5 non-smokers, 21 GOLD 0, 9 GOLD 1, 10 GOLD 2 and 3 GOLD 3 patients). mRNA from the specimens was profiled using Agilent’s Functional ID v2.0 array which contains 23,720 sequences. Quantitative morphometric analysis of the specimens revealed that the samples contained a variable proportion of airways, blood vessels and parenchyma. Incorporating these data into a model relating gene expression to % predicted forced expiratory flow between 25 and 75% of forced expiratory volume (FEF 25-75 % P) revealed a signature gene set of 203 transcripts.

Dataset Information

Rosetta (Merck) Expression Validated Gene Hybridizations

Publications

A comprehensive transcript index of the human genome generated using microarrays and computational approaches.

Similar Datasets

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