Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Stroma+monocytes

ABSTRACT: Gene expression of adherent cells in a coculture of the human bone marrow stromal cell line HS-27a and peripheral blood monocytes (CD14+ cells) was compared to HS-27a cultured alone. Microarray experiments were conducted using CD14+ cells from 7 healthy donors over a period of >7 months. Monocytes were isolated by ficoll separation of whole blood, followed by labelling cells with CD14 monoclonal antibody Tuek4 and magnetic bead selection (Milteny rat anti mouse IgG2a+2b beads) on an AutoMacs. HS27a cells were plated in T75 flasks at approximately 80% confluence. The next day 1.5x10E6 freshly isloated human CD14+ cells were added to the culture. At 3 days the cultures were washed 3x with Hankâ??s buffer to remove nonadherent monocytes. Co-cultures and HS27a cultured alone were harvested by brief trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42ÂºC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS27a and HS27a-monocyte cocultures was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 ul of 3X SSC and 0.8 mg/ml of poly-A for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction and removal of flagged values, log base 2 expression ratios were mean centered and linear transformed to obtain the log and linear values given in the data tables.

ORGANISM(S): Homo sapiens

SUBMITTER: Beverly Torok-Storb

PROVIDER: E-GEOD-831 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Human marrow stromal cells activate monocytes to secrete osteopontin, which down-regulates Notch1 gene expression in CD34+ cells.

Iwata Mineo M Awaya Norihiro N Graf Lynn L Kahl Christoph C Torok-Storb Beverly B

Blood 20040302 12

The hematopoietic microenvironment, approximated in vitro by long-term marrow cultures (LTCs), consists of both nonhematopoietic-derived stromal elements and hematopoietic-derived monocyte/macrophages. To better understand the consequences of monocyte-stroma interactions, we compared gene expression profiles of CD14+ peripheral blood monocytes and HS-27a stromal cells cultured alone and together in cocultures. Results from 7 separate experiments revealed 22 genes were significantly up- or down-r ...[more]

PMID: 14996707

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Project description:Gene expression of adherent cells in a coculture of the human bone marrow stromal cell line HS-27a and peripheral blood monocytes (CD14+ cells) was compared to HS-27a cultured alone. Microarray experiments were conducted using CD14+ cells from 7 healthy donors over a period of >7 months. Monocytes were isolated by ficoll separation of whole blood, followed by labelling cells with CD14 monoclonal antibody Tuek4 and magnetic bead selection (Milteny rat anti mouse IgG2a+2b beads) on an AutoMacs. HS27a cells were plated in T75 flasks at approximately 80% confluence. The next day 1.5x10E6 freshly isloated human CD14+ cells were added to the culture. At 3 days the cultures were washed 3x with Hank’s buffer to remove nonadherent monocytes. Co-cultures and HS27a cultured alone were harvested by brief trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42ºC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS27a and HS27a-monocyte cocultures was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 ul of 3X SSC and 0.8 mg/ml of poly-A for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction and removal of flagged values, log base 2 expression ratios were mean centered and linear transformed to obtain the log and linear values given in the data tables. Keywords: repeat sample

Project description:miRNA expression was compared in skin from control newborn mice and littermate mice ectopically expressing the potent secreted WNT inhibitor Dickkopf 1 (DKK1) in the epidermis. DKK1 completely suppresses hair follicle development. Multiple miRNAs were identified that reproducibly produced signals above background in both types of skin sample. Several miRNAs were identified for which hybridization signals were on average more than 2.5 fold higher in control samples than in Dkk1-expressing samples, suggesting these may be upregulated in hair follicles, and/or are direct or indirect targets of WNT inhibition in the skin. Keywords: miRNA expression array, transgenic mouse, skin, WNT Low molecular weight RNA was isolated using the mirVana RNA extraction kit (Ambion) from full thickness dorsal skin of three K5-rtTA; tetO-Dkk1 newborn mice and three control littermates following doxycycline treatment from E0.5 to induce expression of Dkk1 in double transgenic epidermis [1]. Control and Dkk1âexpressing transgenic samples were tagged for labeling with Cy3 and Cy5 dyes respectively using the Ncode miRNA Labeling System (Invitrogen), mixed by equal mass, and co-hybridized to miRNA arrays that have been described previously [2]. Background-corrected median signals for pixels from each probe spot in both the Cy3 and Cy5 channels were used for analysis. Cy3 (control samples C2, C4, C5) was detected at 532nm and Cy5 (Dkk1âexpressing transgenic samples TG1, TG2, TG4) at 635 nm. References: [1] Chu, E.Y., Hens, J., Andl, T., Kairo, A., Yamaguchi, T.P., Brisken, C., Glick, A., Wysolmerski, J.J., and Millar, S.E. (2004). Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis. Development 131, 4819-4829. [2] Nelson, P.T., Baldwin, D.A., Scearce, L.M., Oberholtzer, J.C., Tobias, J.W., and Mourelatos, Z. (2004). Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1, 155-161.

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Dataset Information

Stroma+monocytes

Publications

Human marrow stromal cells activate monocytes to secrete osteopontin, which down-regulates Notch1 gene expression in CD34+ cells.

Similar Datasets

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