Embryonic stem cell study
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE1310 | GEO | 2004/05/16
SECONDARY ACCESSION(S): PRJNA89907
REPOSITORIES: GEO
Similar Datasets
Project description:Cell culture: H9 hESCs were grown using feeder-free conditions for 7 days after passage then either RNA was harvested or embryoid bodies were prepared, cultured in suspension for 4 days, plated on gelatin-coated plates, and cultured for an additional 9 days in embryoid body medium. Detailed protocols for feeder-free culture and embryoid body formation are available at www.geron.com/scprotocols.pdf RNA preparation: Cells were then harvested in situ by overlaying with RLT lysis buffer (Qiagen, Valencia, CA). RNA was prepared using RNeasy Midi kits as described by the manufacture (Qiagen). To remove contaminants, RNA was precipitated by adding an equal volume of LiCl Precipitation Solution (Ambion, Austin, TX), washed with 70% ethanol and resuspended in water. Probe preparation: Probes were prepared from 20ug of total RNA by direct incorporation of Cy3 or Cy5 dCTP (Amersham Biosciences, Piscataway, NJ) into oligo dT primed first strand cDNA using SuperscriptIII reverse transcriptase (Invitrogen), as described by the dye manufacturer. RNA was removed by alkaline hydrolysis, then neurtralized probes were purified using Qiaquick columns as described by the manufacturer (Qiagen). Hybridization/Washing: Purified probes were dried and resuspended MWG Hybridization Buffer C (MWG Biotech) with the addition of 20 ug of sheared salmon sperm DNA. Slides were hybridized overnight at 42°. After the coverslips were removed, the slides were rinsed in 2X SSC + 0.1% SDS, washed in 2X SSC + 0.1% SDS for 5 min, 1X SSC for 5 min, 0.2X SSC for 5 min and 0.1X SSC for 5 min. Slides were then dried by centrifugation. Scanning: Arrays were scanned using a GenePix 4000A microarray scanner (Axon Insturments, Fremont, CA) with 100% scan power and PMT voltage set to minimize saturated pixels and approximately equalize signal intensities in the two channels. Image processing and data extraction were performed using GenePix Pro 3.0.6 (Axon Instruments). Data Analysis: Average background signal from negative control spots was subtracted from Cy3 and Cy5 signal intensities. In cases where background was larger than signal, signal was set to 1. Log ratios were calculated for each spot and log ratios from replicate spots were averaged. Data was normalized using the âLowessâ method, and analyzed for differential expression. Differentially expressed genes were defined as those with log transformed expression ratios more than 1.5 SD from the mean. This lower threshold was chosen since comparison of the microarray dat to qRT-PCR on the samples used for the microarray experiment indicated that the data was compressed. For example, TDGF1 was up-regulated 100-fold in uES versus EB by qRT-PCR, but only ~5-fold by microarray analysis in the same sample. Normalization and identification of differentially expressed genes was performed using the MIDAS package (Saeed et al. (2003) Biotechniques 34(2):374-8).
2010-06-09 | E-GEOD-1310 | biostudies-arrayexpress
Project description:This series repressents the data set from the paper "Expression microarray analysis and oligo array CGH of aquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations". Biological material: One solid mouse tumor "Colon 26A" was routinely maintained by successive transplantation. A subset of mice with this tumor was treated at the maximum tolerable dosage of Gemcitabine and successively transplanted 5 times and similarly treated. The last passage was completly resistant and designated, "Colon 26G". Expression array: Total RNA was isolated from frozen mouse tumors using Tryzol. Single stranded cDNA was synthetized from 30 mg of total RNA by reverse transcription using aminoallyl labeled dUTP (Ambion). Labelling was performed according to the aminoallyl labeling protocol. Briefly, cDNA was incubated at room temperature for 1 h with fluorolink monofunctional cy5 or cy3 (Amersham) followed by 15 min 4 M hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen) and mixed with 12 mg poly(dA) (Amersham), 60 mg yeast tRNA (Sigma) and 24 mg Cot-1 DNA (Invitroen). The probe was dissolved in 127 ml hybridization mixture containing 46% formamide, 9.5% dextran sulfate, 2x SSC and 0.2% SDS. The probe was heated to 70°C for 10 min and annealed at 37°C for 1 h. Slides were hybridized for 14 h at 37°C over-night (HyArray 12™, Perkin Elmer). After hybridization the slides were washed in the HybArray 12™ with 50% formamide, 2x SSC, pH 7 at 35°C for 15 min, followed b PI buffer (0.1 M sodium phosphate, 0.1% Igepal Ca630, pH 8) at room temperature and 3 washes of 0.2x SSC, 0.1x SSC and 0.01x SSC at room temperature followed by centrifugation. Arrays were scanned using a laser scanner (ScanArray Express, Perkin Elmer) and analysed using ImaGene version 5.6. Cy5 cy3 ratios are calculating by taking the log2 of the signal mean of each spot minus the median background mean of all spots on the array. This is followed by a standard normalization for spot intensity and calculation of the ratios. Keywords: other
2005-12-21 | GSE2352 | GEO
Project description:This series repressents the data set from the paper "Expression microarray analysis and oligo array CGH of aquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations". Biological material: One solid mouse tumor "Colon 26A" was routinely maintained by successive transplantation. A subset of mice with this tumor was treated at the maximum tolerable dosage of Gemcitabine and successively transplanted 5 times and similarly treated. The last passage was completly resistant and designated, "Colon 26G". Expression array: Total RNA was isolated from frozen mouse tumors using Tryzol. Single stranded cDNA was synthetized from 30 mg of total RNA by reverse transcription using aminoallyl labeled dUTP (Ambion). Labelling was performed according to the aminoallyl labeling protocol. Briefly, cDNA was incubated at room temperature for 1 h with fluorolink monofunctional cy5 or cy3 (Amersham) followed by 15 min 4 M hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen) and mixed with 12 mg poly(dA) (Amersham), 60 mg yeast tRNA (Sigma) and 24 mg Cot-1 DNA (Invitroen). The probe was dissolved in 127 ml hybridization mixture containing 46% formamide, 9.5% dextran sulfate, 2x SSC and 0.2% SDS. The probe was heated to 70°C for 10 min and annealed at 37°C for 1 h. Slides were hybridized for 14 h at 37°C over-night (HyArray 12â¢, Perkin Elmer). After hybridization the slides were washed in the HybArray 12⢠with 50% formamide, 2x SSC, pH 7 at 35°C for 15 min, followed b PI buffer (0.1 M sodium phosphate, 0.1% Igepal Ca630, pH 8) at room temperature and 3 washes of 0.2x SSC, 0.1x SSC and 0.01x SSC at room temperature followed by centrifugation. Arrays were scanned using a laser scanner (ScanArray Express, Perkin Elmer) and analysed using ImaGene version 5.6. Cy5 cy3 ratios are calculating by taking the log2 of the signal mean of each spot minus the median background mean of all spots on the array. This is followed by a standard normalization for spot intensity and calculation of the ratios.
2010-06-05 | E-GEOD-2352 | biostudies-arrayexpress
Project description:Human hepatocellular carcinoma cells, HepG2 , cultured in DMEM containing 10% fetal calf serum. To study gene expression changes induced by Vitamin K2, total RNA was isolated following the Isogen procedure (Nippon-gene, Tokyo, Japan) from HepG2 cells with and without 50 microM of Vitamin K2 treatment for three days. The cDNA microarrays Human Oligo Chip 30K “AceGene” subset A was purchased from Hitachi Software Engineering Co. (Yokohama-City, Japan). This array contains 10368 kinds of gene-specific 50 mer sense oligonucleotides. Subset A contains mainly known function ORF oligo probes. Preparation of fluorescent cDNA targets by an indirect labeling approach was performed using PowerScript reverse transcription kit (Clontech). First-strand cDNAs were synthesized from 30 microg of total RNA from HepG2 cells treated with or without Vitamin K2 using random primer and PoweScript Fluorescent Labelling Kit. Monofunctional, N-hydroxysuccinimide-activated fluorescent dyes (Cy3 and Cy5; Amersham) were coupled to the cDNAs by reaction with the amino functional groups. Untreated cells were labeled with Cy3 as a control and treated cells with Cy5 as a sample. After free dyes were removed using MinElute Purification kit (Qiagen), the targets were mixed together and added to the microarrays, and then incubated overnight (16 hours) at 42?C. The slides were washed at 30?C in each with 2x SSC, 0.1% SDS and 2x SSC for 10 min and 5 min, respectively, and washed in 1x SSC for 5 min. Fluorescent images of the hybridized microarrays were scanned with a fluorescence laser confocal slide scanner (Affymetrix 428 Array Scanner, Affymetrix, Santa Clara, CA). The Cy3 and Cy5 intensities with background subtraction were determined by ImaGene 4.2 software (BioDiscovery, Marina Del Rey, CA). The expression data were then filtered based on their channel intensities, spot size and flag (missing or inaccurate data), and the Cy5/Cy3 ratios were calculated and normalized by median-centering the log-ratio of all genes.
2010-12-01 | GSE25762 | GEO
Project description:A single-domain globin (cgb) mutant strain of Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 10 uM NOC-5 & -7 (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Keywords: Stress-response of a cgb mutant to NO A C. jejuni NCTC11168 cgb mutant was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 μl sample onto both nutrient agar plates incubated at 37 °C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 °C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, NOC-5 & -7 (final concentration 10 uM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. cDNA synthesis was carried out using 12 µg of starting material, which was primed with 9 µg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 µl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 °C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 °C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 °C. Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 °C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of ≤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed
2007-12-31 | E-GEOD-5433 | biostudies-arrayexpress
Project description:A single-domain globin (cgb) mutant strain of Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 0.25 mM GSNO (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagenâ??s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. A C. jejuni cgb mutant was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 μl sample onto both nutrient agar plates incubated at 37 °C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 °C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, GSNO (final concentration 0.25 mM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. cDNA synthesis was carried out using 12 µg of starting material, which was primed with 9 µg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 µl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 °C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 °C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 °C. Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 °C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â?¥ twofold and displayed and P value of â?¤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-30 | E-GEOD-5396 | biostudies-arrayexpress
Project description:Wild-type Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 10 uM NOC-5 & -7 (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagenâ??s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. C. jejuni NCTC11168 was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 μl sample onto both nutrient agar plates incubated at 37 °C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 °C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, NOC-5 & -7 (final concentration 10 uM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. cDNA synthesis was carried out using 12 µg of starting material, which was primed with 9 µg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 µl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 °C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 °C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 °C. Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 °C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â?¥ twofold and displayed and P value of â?¤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-30 | E-GEOD-5400 | biostudies-arrayexpress
Project description:Batch cultures of Wild-type C. jejuni NCTC11168 were grown in 150 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % O2, 10 % CO2, 80 % N2) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ºC. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 10 minute exposure samples of both treated and untreated cells were harvested into phenol/ethenol to stabilize the RNA and total RNA was purified using Qiagenâs RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. A C. jejuni cgb mutant was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 μl sample onto both nutrient agar plates incubated at 37 °C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 °C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, GSNO (final concentration 0.25 mM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. cDNA synthesis was carried out using 12 µg of starting material, which was primed with 9 µg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 µl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 °C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 °C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 °C. Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 °C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ⥠twofold and displayed and P value of ⤠0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-30 | E-GEOD-5397 | biostudies-arrayexpress
Project description:Two batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 100 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached 0.010 mM NOC-5 & NOC-7 was added to one of the cultures. After a 15 minute exposure samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using QiagenM-bM-^@M-^Ys RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 100 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 10 minute exposure, 30 ml samples of both treated and untreated cells were mixed immediately on ice with 3.56 ml 100% ethanol and 185 M-BM-5l phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. Equivalent amounts of RNA (15 M-NM-<g) from control and test cultures were used as template for synthesis of labelled cDNA. Labelling was done by using dCTP nucleotide analogues containing either Cy3 or Cy5 fluorescent dyes. RNA was primed with 9 M-NM-<g pd(N)6 random hexamers (Amersham Biosciences). For annealing, the mixture was incubated for 10 min at 65oC and then 10 min at room temperature. Each reaction mixture (0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP, 0.1 mM DTT (Invitrogen) and 1 mM Cy3-dCTP or Cy5-dCTP, total volume 25ul) was incubated for 3 h at 42 oC with 200 U of Superscript III RNase-H Reverse Transcriptase (Invitrogen). The reaction was terminated by the addition of 5u1 mM NaOH and heating the tube to 65 oC for 10 min to hydrolyse the RNA. Then it was neutralised with 5ul 1M HCl and 1 M TE (pH 8). Purification of cDNA was done with a PCR purification kit (Qiagen). The cDNA was eluted and resuspended in 30 M-NM-<l elution buffer (Qiagen, supplied in kit). Each slide set (control slide and dye-swap) was prepared as follows: For control slide Cy3-dCTP labelled control cDNA was mixed with Cy5-dCTP labelled test cDNA. For dye-swap slide Cy5-dCTP labelled control cDNA was mixed with Cy3-dCTP labelled test cDNA. This is made for compensate possible differences in the labelled nucleotides incorporation. The slides used were C. jejuni OciChipM-BM-. arrays from Ocimum Biosolutions. The cDNA mixture for each slide was dried by evaporation for approximately 35 min in a SPD 121P SpeedVacM-BM-. (Thermo Savant, Waltham, MA, USA) The dry cDNA was resuspended in pre-warmed (42M-BM-:C) salt-based hybridisation buffer (Ocimum Biosolutions) and was heated to 95 M-BM-0C for 3 min and then placed on ice for 3 min. The spoted area of the slide (located with an array finder) was enclosed within a gene frame (MWG/Ocimum). The cDNA suspension was distributed through the inner space of the gene frame and enclosed with an air-tight coverslip. The slides were incubated for 16-24 hours at 42M-BM-: C in sealed MWG hybridisation chambers shaken in a water bath. After incubation, gene frames and coverslips were removed and slides washed sequentially in 2x, 1x, 0.2x y 0.1x SSC buffer, by shaking for 5 minutes at 80 rpm at 37M-BM-: C pre-warmed buffer. (2x buffer was supplemented with 1% SDS). Then the slides were dried by centrifugation at 250 x g for 5 min. Slides were scanned using an Affymetrix 428 scanner. The processing of images and quantification of the microarrays signal was done using software from Biodiscovery Inc (Imagene, version 4.0 and Genesight, version 4.0). Spots with signal intensity lower than background or other significant blemishes were eliminated from subsequent processing. Mean values from each channel were then log2 transformed and normalised using the Subtract by Mean method to remove intensity-dependent effects in the log2 (ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalised values. Significance analysis of the data used the StudentM-bM-^@M-^Ys t test to determine the probability that the average of the experimental replicates was significantly different from the average of the control replicates. p-values for the data were calculated by treating each slide as a repeat using Genesight 4. Genes differentially regulated M-bM-^IM-% 2-fold and displaying a p-value of M-bM-^IM-$ 0.05 were defined as being statistically significant and differentially transcribed.
2012-05-22 | E-GEOD-38114 | biostudies-arrayexpress
Project description:Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached 0.010 mM NOC-5 & NOC-7 was added to one of the cultures. After a 15 minute exposure samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using QiagenM-bM-^@M-^Ys RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 M-BM-:C. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 10 minute exposure, 30 ml samples of both treated and untreated cells were mixed immediately on ice with 3.56 ml 100% ethanol and 185 M-BM-5l phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. Equivalent amounts of RNA (15 M-NM-<g) from control and test cultures were used as template for synthesis of labelled cDNA. Labelling was done by using dCTP nucleotide analogues containing either Cy3 or Cy5 fluorescent dyes. RNA was primed with 9 M-NM-<g pd(N)6 random hexamers (Amersham Biosciences). For annealing, the mixture was incubated for 10 min at 65oC and then 10 min at room temperature. Each reaction mixture (0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP, 0.1 mM DTT (Invitrogen) and 1 mM Cy3-dCTP or Cy5-dCTP, total volume 25ul) was incubated for 3 h at 42 oC with 200 U of Superscript III RNase-H Reverse Transcriptase (Invitrogen). The reaction was terminated by the addition of 5ul 1 mM NaOH and heating the tube to 65 oC for 10 min to hydrolyse the RNA. Then it was neutralised with 5ul 1 M HCl and 1 M TE (pH 8). Purification of cDNA was done with a PCR purification kit (Qiagen). The cDNA was eluted and resuspended in 30 M-NM-<l elution buffer (Qiagen, supplied in kit). Each slide set (control slide and dye-swap) was prepared as follows: For control slide Cy3-dCTP labelled control cDNA was mixed with Cy5-dCTP labelled test cDNA. For dye-swap slide Cy5-dCTP labelled control cDNA was mixed with Cy3-dCTP labelled test cDNA. This is made for compensate possible differences in the labelled nucleotides incorporation. The slides used were C. jejuni OciChipM-BM-. arrays from Ocimum Biosolutions. The cDNA mixture for each slide was dried by evaporation for approximately 35 min in a SPD 121P SpeedVacM-BM-. (Thermo Savant, Waltham, MA, USA) The dry cDNA was resuspended in pre-warmed (42M-BM-:C) salt-based hybridisation buffer (Ocimum Biosolutions) and was heated to 95 M-BM-0C for 3 min and then placed on ice for 3 min. The spoted area of the slide (located with an array finder) was enclosed within a gene frame (MWG/Ocimum). The cDNA suspension was distributed through the inner space of the gene frame and enclosed with an air-tight coverslip. The slides were incubated for 16-24 hours at 42M-BM-: C in sealed MWG hybridisation chambers shaken in a water bath. After incubation, gene frames and coverslips were removed and slides washed sequentially in 2x, 1x, 0.2x y 0.1x SSC buffer, by shaking for 5 minutes at 80 rpm at 37M-BM-: C pre-warmed buffer. (2x buffer was supplemented with 1% SDS). Then the slides were dried by centrifugation at 250 x g for 5 min. Slides were scanned using an Affymetrix 428 scanner. The processing of images and quantification of the microarrays signal was done using software from Biodiscovery Inc (Imagene, version 4.0 and Genesight, version 4.0). Spots with signal intensity lower than background or other significant blemishes were eliminated from subsequent processing. Mean values from each channel were then log2 transformed and normalised using the Subtract by Mean method to remove intensity-dependent effects in the log2 (ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalised values. Significance analysis of the data used the StudentM-bM-^@M-^Ys t test to determine the probability that the average of the experimental replicates was significantly different from the average of the control replicates. p-values for the data were calculated by treating each slide as a repeat using Genesight 4. Genes differentially regulated M-bM-^IM-% 2-fold and displaying a p-value of M-bM-^IM-$ 0.05 were defined as being statistically significant and differentially transcribed.
2012-05-22 | E-GEOD-38115 | biostudies-arrayexpress