Gis2 mutant versus wild-type cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE24004 | GEO | 2010/12/22
SECONDARY ACCESSION(S): PRJNA133215
REPOSITORIES: GEO
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Project description:To measure the relative changes of gene expression upon Gis2p depletion, 50 ml of delta Gis2 cells or the respective wild-type strain BY4741 were grown in YPD at 30 degrees celsius to an OD600 of 0.5- 0.6. Cells were harvested by centrifugation and washed twice with 5ml of bidistilled water (ddH2O). RNA was isolated by hot phenol extraction for microarray analysis. 20 ug of total RNA derived from wild-type and knock-out cells were reverse transcribed in the presence of 5-(3-aminoallyl)-dUTP and natural dNTPs with a mixture of N9 and dT20V primers, and cDNAs were covalently linked to Cy3 and Cy5 NHS-monoesters (GE HealthSciences Cat# RPN5661), respectively, and competitively hybridized onto yeast oligo arrays at 42 degrees celsius for 14 hours in formamide-based hybridization buffer. Microarrays were scanned with an Axonscanner 4200 (Molecular Devices) and analyzed with GenePix Pro 5.1 (Molecular devices) before uploading into Stanford Microarray Database. Biological Replicate
2010-12-22 | E-GEOD-24004 | biostudies-arrayexpress
Project description:Measure the relative changes of gene expression upon GIS2 overexpression; 100 ml of BY4741 cells bearing plasmid pBG1805-Gis2 (GIS2 under the control of the galactose promotor) or the empty plasmid pBG1805 (=control) were grown in synthetic medium lacking uracil (SC-Ura supplemented with 2% raffinose) at 30 degrees to an OD600 of 0.45- 0.5. Expression of GIS2 was induced with 2% galactose for 1.5 h and cells were harvested by centrifugation, washed twice with 800 ul of ice-cold sterile water. 100 ul (1/8) of cells were removed and RNA was isolated by hot phenol extraction for microarray analysis. 6.7 ug of total RNA derived from cells expressing the empty vector (pBG1805) or Gis2p (pBG1805-Gis) were reverse transcribed in the presence of 5-(3-aminoallyl)-dUTP and natural dNTPs with a mixture of N9 and dT20V primers, and cDNAs were covalently linked to Cy3 and Cy5 NHS-monoesters (GE HealthSciences Cat# RPN5661), respectively, and competitively hybridized on yeast oligo arrays at 42 degrees celsius for 14 hours in formamide-based hybridization buffer. Microarrays were scanned with an Axonscanner 4200 (Molecular Devices) and analyzed with GenePix Pro 5.1 (Molecular devices).
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Project description:Measure the relative changes of gene expression upon GIS2 overexpression; 100 ml of BY4741 cells bearing plasmid pBG1805-Gis2 (GIS2 under the control of the galactose promotor) or the empty plasmid pBG1805 (=control) were grown in synthetic medium lacking uracil (SC-Ura supplemented with 2% raffinose) at 30 degrees to an OD600 of 0.45- 0.5. Expression of GIS2 was induced with 2% galactose for 1.5 h and cells were harvested by centrifugation, washed twice with 800 ul of ice-cold sterile water. 100 ul (1/8) of cells were removed and RNA was isolated by hot phenol extraction for microarray analysis. 6.7 ug of total RNA derived from cells expressing the empty vector (pBG1805) or Gis2p (pBG1805-Gis) were reverse transcribed in the presence of 5-(3-aminoallyl)-dUTP and natural dNTPs with a mixture of N9 and dT20V primers, and cDNAs were covalently linked to Cy3 and Cy5 NHS-monoesters (GE HealthSciences Cat# RPN5661), respectively, and competitively hybridized on yeast oligo arrays at 42 degrees celsius for 14 hours in formamide-based hybridization buffer. Microarrays were scanned with an Axonscanner 4200 (Molecular Devices) and analyzed with GenePix Pro 5.1 (Molecular devices). Biological Replicate
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Project description:Measure the relative changes of gene expression upon GIS2 overexpression; BY4741 cells bearing plasmid pBG1805-Slf1 or control plasmid pBG1805 were grown in SC ura- gal at 30 C for 6 or 24 hours. An overnight culture of cells was inoculated at an OD600 of 0.1 and further grown to an OD600 of 0.5- 0.7 in 60 ml SC Ura- Gal at 30 C. Cells were harvested by centrifugation, washed twice with 600 ul of ice-cold sterile water. 200 ul of cells were removed and RNA was isolated by hot phenol extraction for microarray analysis. 7.5 ug of total RNA derived from cells expressing the empty vector (pBG1805) or Slf1p (pBG1805_Slf1) were reverse transcribed in the presence of 5-(3-aminoallyl)-dUTP and natural dNTPs with a mixture of N9 and dT20V primers, and cDNAs were covalently linked to Cy3 and Cy5 NHS-monoesters (GE HealthSciences Cat# RPN5661), respectively, and competitively hybridized on yeast oligo arrays at 42 degrees celsius for 14 hours in formamide-based hybridization buffer. Microarrays were scanned with an Axonscanner 4200 (Molecular Devices) and analyzed with GenePix Pro 5.1 (Molecular devices). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
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Project description:To compare total RNA levels in miR-124 and mock-transfected cells (Figure S3), 5-10 ug of total RNA from miR-124-transfected cells or mock-transfected cells or universal reference RNA (Stratagene Cat# 740000) was reverse transcribed with Superscript III (Invitrogen Cat# 18080085) in the presence of aminoallul-dUTP 5-(3-aminoallyl)-dUTP (Ambion Cat# AM8439) and natural dNTPs (GE Healthsciences Cat# US77212) with 10 ug of N9 primer (Invitrogen). Subsequently, amino-allyl-containing cDNAs from miR-124 and mock-transfected cells were covalently linked to Cy5 NHS-monoesters, and universal reference cDNA was covalently linked to Cy3 NHS-monoesters (GE HealthSciences Cat# RPN5661). Cy5- and Cy3-labeled cDNAs were mixed and diluted into 50 ul of solution containing 3x SSC, 25 mM Hepes-NaOH (pH 7.0), 20 ug human Cot-1 DNA (Invitrogen Cat# 15279011), 20 ug of poly(A) RNA (Sigma Cat# P9403), 25 ug of yeast tRNA (Invitrogen Cat# 15401029), and 0.3% SDS. The sample was incubated at 95 C for 2 min, spun at 14,000 rpm for 10 mins in a microcentrifuge, then hybridized at 65 C An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
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