Dataset Information

Comparison between Saccharomyces cerevisiae HS 10, HS 34 and HS 12, HS 37

ABSTRACT: Gene expression of different flocculent (HS 10 and HS 34) and powdery (HS 12 and HS 37) yeast strains compared to each other during exponential and stationary growth phase was analysed. The isolation of RNA was done by disruption of the cells under liquid nitrogen using mortar and pistil and then the Qiagen RNeasy Midi Kit with some modifications within the manufacturers´ protocol and the Qiagen RNase-Free DNase Set were applied. 6 µg of the total RNA per sample was used for each microarray experiments. The indirect labelling by the tyramide-signal-amplification method (MicromaxTM TSATM labelling and detection Kit from Perkin Elmer life sciences) was used to increase the Cy3 and Cy5 signals of microarray detection. Each cDNA containing Biotin- and Fluorescein-nucleotides respectively was purified with a QIAquick PCR purification kit and suspended in 11 µl of the formamide containing hybridization buffer. The slides were hybridized at 42°C over night under a cover slip. The microarrays were scanned by the Axon 4000B scanner; image intensity data were extracted and analysed with GenePix® Pro 6.0 software. Data from different scans of Dye-swap experiment were extracted by GenePix Pro 6.0 software, normalized and united. An outliertest has been applied in order to find outliers amongst the gene replicats. Subsequently, a t-test (1% and 5% probability of error) has been used in order to find regulated genes. Keywords: sorted yeast cells

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE12369 | GEO | 2008/12/12

SECONDARY ACCESSION(S): PRJNA113201

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

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:The diagnosis of cerebral vasospasm after Subarachnoid-Hemorrhage is currently very difficult, additional tools such as blood biomarkers are necessary. We tested the ability of gene expression profiles of blood cells to predict vasospasm. 32 patients suffering subarachnoid-hemorrhage were included in this prospective monocentre study. They were grouped according to have a complicated cerebral vasospasm (Vasospasm) or not (Control) and Paired according to age (+/- 10 years), sex, Fisher grade (+/- 1), location, smoking (at least 3 first parameters). Gene expression profiles of blood cells were determined using 25,000~gene microarray. Blood sample: 2.5 mL harvested in PAXgene® Blood RNA tubes (PreAnalytix) RNA extraction: PAXgene® Blood RNA kit (Qiagen). We used a Universel Reference RNA (Stratagene). RNA amplification and labelling: kit Amino Allyl MessageAmp II (Ambion). We hybridized 4 microarrays per patient using pangenomic microarrays from the "Réseau National des Génopôles" (Illkirch, France). 2 slides were hybridized with reference RNA labelled Cy3 and patient RNA labelled Cy5, and 2 slides were hybridized with reference RNA labelled Cy5 and patient RNA labelled Cy3. Hybridation : Agilent protocol with few modifications : 750 ng of each labelled RNA were hubriddized at 60°C during 17 hours in an Aglient hybridization oven. After washings, Slides were scanned with a GenePix 4000B scanner (Molecular Devices). Image intensity data were extracted with GenePix Pro 6.0 analysis software. Quantification of Cy3 and Cy5 and selection of good spots were performed using the MAIA software (Novikov E and Barillot E. Software package for automatic microarray image analysis (MAIA). The ACUITY software was then used to normalize log ratios Cy3/Cy5 with Lowess non linear normalization, to filter out genes not present in at least 3 slides out of 4, to evaluate the reproducibility of the 4 microarrays of each patient (hierarchical clustering, Self Organizing Maps). Statistical analyses to insure reproducibility was performed using Excel (correlation coefficients, ANOVA). Only slides that passed all reprocubility tests were validated.

Project description:Study of the relation between the activation of the inflammation in the encephalic death donor and the quality of the graft (time before the first rejection occurred). Diagnostic approach based on the profiling of the donor's inflammatory reaction. Keywords: procalcitonin, graft, inflammation The 22 patients of this study were divided in 2 groups corresponding to the extreme quartiles of PCT values. The first group are the patients with low PCT and the second group are the patients with high PCT. Blood sample: 2.5 mL harvested in PAXgene® Blood RNA tubes (PreAnalytix) RNA extraction: PAXgene® Blood RNA kit (Qiagen) We used a Universel Reference RNA (Stratagene) RNA amplification and labelling: kit Amino Allyl MessageAmp II (Ambion) We hybridized 4 microarrays per patient using pangenomic microarrays from the "Réseau National des Génopôles" (Illkirch, France). 2 slides were hybridized with reference RNA labelled Cy3 and patient RNA labelled Cy5, and 2 slides were hybridized with reference RNA labelled Cy5 and patient RNA labelled Cy3. Hybridation : Agilent protocol with few modifications : 750 ng of each labelled RNA were hubriddized at 60°C during 17 hours in an Aglient hybridization oven. After washings, Slides were scanned with a GenePix 4000B scanner (Molecular Devices). Image intensity data were extracted with GenePix Pro 6.0 analysis software. Quantification of Cy3 and Cy5 and selection of good spots were performed using the MAIA software (Novikov E and Barillot E. Software package for automatic microarray image analysis (MAIA). Bioinformatics. 2007 Mar 1;23(5):639-40. The ACUITY software was then used to normalize log ratios Cy3/Cy5 with Lowess non linear normalization, to filter out genes not present in at least 3 slides out of 4, to evaluate the reproducibility of the 4 microarrays of each patient (hierarchical clustering, Self Organizing Maps). Statistical analyses to insure reproducibility was performed using Excel (correlation coefficients, ANOVA). Only slides that passed all reprocubility tests were validated.

Dataset Information

Comparison between Saccharomyces cerevisiae HS 10, HS 34 and HS 12, HS 37

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets