title: HUC Cells grown on SISgel, Replicate 1

description:
RNA was isolated from the cells growing on SISgel using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background 37893.95 +/- 3328.56
Keywords = bladder cancer

source_name: HUC Cells grown on SISgel


title: HUC cells on Matrigel, Replicate 1

status: Public on Nov 02 2003

type: RNA

city: Oklahoma City

first: Robert

middle: Evan

last: Hurst

state: OK

address: 920 S. L. Young Blvd

department: Urology

country: USA

institute: Oklahoma University Health Sciences Center

description:
RNA was isolated from the cells growing on SISgel using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 37059.30 +/- 3418.05
Keywords = Bladder cancer

molecule: total RNA

organism: Homo sapiens

source_name: HUC cells grown on Matrigel


title: J82 Cells grown on plastic, Replicate 1

description:
RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 120,626.9 +/- 18183.68
Keywords = Bladder cancer

source_name: J82 cells grown on plastic


title: J82 Cells grown on plastic, Replicate 2

description:
RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 4670.98 +/- 4322.65
Keywords = Bladder cancer


title: TCCSUP cells grown on plastic, Replicate 2

description: RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 39,964.66 +/- 4904.91

source_name: TCCSUP cells grown on plastic


title: JBV Cells grown on plastic, Replicate 1

description:
RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean Background = 31,886.11 +/- 2462.39
Keywords = Bladder cancer

source_name: JBV Cells grown on plastic


title: JP 253 Cells grown on plastic, Replicate 2

description:
RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 38022.88 +/- 2483.17
Keywords = Bladder cancer

source_name: JP 253 cells grown on plastic


title: HUC Cells grown on SISgel, Replicate 2

description:
RNA was isolated from the cells growing on SISgel using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 36869.25 +/- 2350.92
Keywords = Bladder cancer


title: TCCSUP cells grown on SISgel, Replicate 1

description:
RNA was isolated from the cells growing on SISgel or Matrigel using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 36649.15 +/- 2086.23
Keywords = Bladder cancer

source_name: TCCSUP cells grown on SISgel


title: TCCSUP cells grown on plastic, Replicate 1

description:
RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 31,581.15 +/- 2404.66
Keywords = Bladder cancer


title: JBV Cells grown on plastic, replicate 2

description: RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 38,129.32 +/- 2355.37


title: HUC Cells on Plastic Replicate 2

description: RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 49,277.53 +/- 4387.11

source_name: HUC Cells on Plastic


title: JP 253 Cells grown on plastic, Replicate 1

description:
RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel. The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column. Yield of total RNA and the purity were assessed by spectrophotometry. The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter. First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied. The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step. A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system. Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide. After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company. The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software. The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 50,054.80 +/- 4605.49
Keywords = bladder cancer

source_name: JP 253 grown on plastic


title: ACX018

status: Public on Mar 25 2003

city: Evry

first: Jerome

last: Lamartine

address: 2 rue Gaston Cremieux

department: Departement de Radiobiologie et Radiopathologie

country: France

institute: CEA

description:
HaCaT keratinocytes were seeded at 10 000 cells /cm2 and cultured as monolayers in DMEM supplemented with 10% fetal calf serum at 37°C and 5% CO2. At day 7, the culture was confluent, and it was further kept in culture for 3 days. In these conditions, cell density-arrested keratinocytes undergo the differentiation program found in the suprabasal layers of the epidermis (spinous and granular layers).
For the microarray studies, 300 cm2 flasks were irradiated at day 3 and 10 with a 60cobalt source at a dose rate of 0,4 Gy/min. A radiation dose of 2 Gy was used. Cells were incubated again after exposure, and cells were harvested at 24 hours after irradiation. Reference cells followed exactly the same treatment but were not irradiated.
Total RNA was isolated using the Tripur reagent (Roche Molecular Biochemicals, Meylan, France) following the manufacturer’s instruction and dissolved in RNAse-free water. The quality of RNA was controlled by using an Agilent 2100 Bioanalyser (Agilent technologies, Palo Alto, CA, USA) and concentration was measured by absorbance at 260 nm.
Labelling of total RNA was performed by incorporation of aminoallyl-modified dUTP (Sigma, St Quentin-Fallavier, France), followed by a subsequent incorporation of monofunctionnal Cy3 or Cy5-N-succinimide esters (Amersham Biosciences, Orsay, France). For each labelling experiment, 20 µg of total RNA was used in an oligo(dT) primed reaction in the presence of 200 µM aminoallyl-dUTP, 500 µM each dATP, dCTP, dGTP, 300 µM dTTP, 400 U of Superscript II polymerase (Invitrogen, Cergy Pontoise, France) in a final volume of 30 µl. After incubation at 42°C for 2 hours, RNA was hydrolyzed by adding 2 units of DNAse-free RNAse (Life Technologies) and incubation at 37°C for 15 minutes. The volume was adjusted to 500 µl with H20, aminoallyl-cDNA was purified by centrifugation in a microcon (YM-30 Millipore Amicon), eluted with 40 µl of TE and then dried in a vacuum.
Samples were then resuspended in 10 µl sodium bicarbonate 0.1 M and mixed with monofunctional dye. Coupling reaction was performed during 30 minutes at room temperature in dark.
The Cy3 and Cy5-labeled cDNAs were purified using a Microcon YM30, eluted with 100 µl of TE, mixed with 10 µg of human Cot-1 DNA, 10µg poly(A) and 10µg of yeast tRNA, then precipitated with 0.5 volume of 7.5 M sodium acetate (pH 5.2) in ethanol. The pellet was dissolved in 15 µl of hybridization solution (52% formamide, 1,75x denhardt’s, 0,35% SDS and 6x SSPE). The probe was heated for 2 min at 100°C, incubated at 37°C for 20-30 min and placed between slide and coverslip. The arrays were incubated overnight at 42°C in an humidified slide chamber (Corning) and washed in a first bath of 0.1x SSC with 0.1% SDS for 15 minutes followed by two washing steps in 0.1x SSC for 15 min each at room temperature. The arrays were dried by centrifugation at 600 rpm for 6 min.
Fluorescence intensities of Cy3 and Cy5 were measured separately at 532 and 635 nm with a laser scanner (Axon Genepix4000B).

Keywords = human keratinocytes, gamma radiations

source_name: HaCaT keratinocytes 2 Gy 24h

source_name: HaCaT keratinocytes 0 Gy 24h (reference)


title: Leg Discs iso4 lm50

status: Public on Jan 21 2003

city: Basel

first: Lydia

last: Michaut

address: Klingelbergstrasse 70

department: Cell Biology

country: Switzerland

institute: Biozentrum

description: To analyze gene expression in control leg imaginal discs (iso4 third instar wandering larvae), one to two hundred leg imaginal discs were dissected and immediately transferred into 800ul Trizol (GIBCO Life Technologies)  and stored at –70°C until RNA extraction, which was performed according to the manufacturer’s instructions. Quantity and quality of total RNA were determined by capillary electrophoresis on a RNA6000 bioanalyzer (Agilent Technologies). Biotinylated RNA targets were prepared from 15 to 20 µg total RNA (500-700 leg discs) according to the standard Affymetrix procedure (see Michaut et al., 2003).

organism: Drosophila melanogaster

source_name: Drosophila wandering larvae leg imaginal discs


title: SGlate10

status: Public on Jul 01 2002

first: Boris

last: Egger

state: BS

address: Klingelbergstrasse 50

department: University of Basel

institute: Institute of Zoology

description:
Experimental Design
To identify gcm downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays to analyse differential gene expression in Drosophila wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm.  The wild-type was Oregon-R.  Tissue-specific misexpression was achieved by using virgin females from scabrous-GAL4 (Klaes et al., 1994) that were crossed to yw; UAS-gcm; UAS-gcm males (Jones et al., 1995).  Transcripts were analysed at two defined temporal windows during embryogenesis.  First, during a period of initial gcm action on determination of glial cell precursors and second during a later period when glial cells have already differentiated.  After a 1 hr pre-collection, wild-type and sca-gcm embryos were collected in parallel for 1 hr and staged to 6-7 hrs AEL (stage 11) or to 13-14 hrs AEL (late stage 15/early stage 16).  Stages are according to (Campos-Ortega and Hartenstein, 1997).

Samples used, extract preparation and labeling
Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast-prep homogenizer FP120 (BIO101).  After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech).  cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.  This primer (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3’) was PAGE purified.  For first-strand cDNA synthesis, a typical 20 μl reaction contained 20 μg total RNA, 100 pmol T7-(T)24 primer, 1 mM of each dNTP and 800 units reverse transcriptase (AMV Superscript II).  The reaction was incubated for 1 hr at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 hrs in a total volume of 150 μl, using 10 units E. coli DNA ligase, 40 units E. coli DNA polymerase I and 4 units Rnase A in the presence of 200 μM dNTPs.  After second-strand cDNA synthesis, samples were incubated with 20 units T4 DNA polymerase at 18°C for 5 min.  Then, 0.5 μl Rnase A 100 (μg/μl) (Quiagen) was added and the samples were incubated at 37°C for 30 min.  Thereafter, 2.5 μl proteinase K (30 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min.  After cDNA synthesis was completed, the samples were phenol-chloroform extracted using Phase Lock Gel (Eppendorf) and ethanol precipitated.  Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit, Ambion).  A 20 μl reaction volume contained between 0.3-1.5 μg cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2μl 10x T7 enzyme mix.  The reaction was incubated at 37°C for 6-8 hrs.  Thereafter, the unincorporated dNTPs were removed by running the sample over an Rneasy spin column (Qiagen).  Samples were precipitated, dissolved in 20 μl DEPC-treated water and spectrophotometrically quantified.  Thereafter, 35 μg of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 45 min in a volume of 25 μl, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate.  After fragmentation, the samples were placed on ice.

Hybridization procedures
Gene Chips were prehybridized with 220 μl hybridization buffer ( 1x MES (pH 6.7), 1M NaCl, 0.01 % Tween 20, 0.5 μg/μl acetylated BSA, 0.5 μg/μl sonicated herring sperm DNA ) for 15 min at 40°C on a REAX 2 rotisserie (Heidolph, Swabach, Germany) at 60 rpm.  Hybridization was done in a final volume of 220 μl hybridization buffer containing 35 μg fragmented biotinylated cRNA.  The samples were heated to 95°C for 5 min and briefly spun down.  Hybridization was carried out for 16 hrs at 45°C on a rotisserie at 60 rpm.  After hybridization, the arrays were briefly rinsed with 6x SSPE-Tw (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01 % Tween 20) and washed on a Fluidics Station (Roche) for 5 min.  Chips were rinsed with MES-wash buffer (1x MES, 26 mM NaCl, 0.01 % Tween) and then 230 μl fresh MES-wash-buffer was applied to wash the chips at 45°C for 30 min with rotation.  Hybridized arrays were stained with 230 μl stain buffer (1xMES, 1M NaCl, with  2.3 μl streptavidin-R phycoerithrin conjugate (1 mg/ml) (Molecular Probes) and 2.5 μg/ml acetylated BSA (Sigma) at 40°C for 15 min and at 60 rpm.  The chips were rinsed and washed again on a Fluidics Station (Roche) for 5 min before scanning.

Measurement data and data processing
Probe arrays were scanned with a commercial confocal laser scanner (Hewlett-Packard). Pixel intensities were measured, and expression signals were analysed with commercial software (Genechip 4.0, Affymetrix).

source_name: sca-gcm embryos (13-14 hrs AEL)


title: HUC Cells on Plastic Replicate 1

description:
RNA was isolated from the cells growing on plastic using the QIAeasy kit (Qiagen) by adding 300 microliter lysis buffer and pipeting up and down to lyse the cells and dissolve the gel.  The RNA was isolated from the lysate by use of a QIAshredder spin column to complete homogenization, proteinase K digestion, ethanol precipitation, an RNeasy mini spin column, DNase I digestion, and a final spin column.  Yield of total RNA and the purity were assessed by spectrophotometry.  The yield was approximately 2.4 - 6 microg RNA per culture in 30 microliter.  First strand synthesis was carried out at 42 C for 1 h. with reagents supplied by Clontech as part of the SMART II kit except that Superscript II (Invitrogen) reverse transcriptase (a version of MMLV reverse transcriptase engineered for increased stability) was substituted for the one supplied.  The SMART II oligonucleotide is included to capture full length cDNA at the 5’end and to permit amplification in the next step.  A proportional amplification (2000 to 5000 fold) was then used to produce sufficient probe for analysis using the Clontech SMART (Switch Mechanism At the 5' end of Reverse Transcript) system.  Labeled probe is produced from an aliquot of the amplified probe with Klenow fragment and 32P-labeled nucleotide.  After probe purification, 15 × 10**6 cpm of probe is hybridized in duplicate to the Clontech Human Cancer 1.2 array on nylon following the protocol provided by the company.  The results are visualized by exposing a phosphorimager screen approximately 16 hours overnight and then reading with the Packard Cyclone phosphoimager and Optiquant software.  The background was subtracted from each of the densitometer counts by iteratively eliminating all points that are over 3 standard deviations above the mean of the 280 background measurements provided for each array. Mean background = 41,766.89 +/- 2793.35
Keywords = Bladder cancer


title: WTlate04

source_name: wild-type embryos (13-14 hrs AEL)


title: WTlate01


title: SGlate08


title: WTlate02


title: Leg Discs iso4 lm49


title: Eye Discs iso4 lm18

status: Public on Jan 08 2003

description: To analyze gene expression in Drosophila eye primordia (third instar wandering larvae), the eye part was separated from the antennal part of the eye-antennal disc. One hundred eye imaginal discs were dissected and immediately transferred into 800ul Trizol (GIBCO Life Technologies)  and stored at –70°C until RNA extraction, which was performed according to the manufacturer’s instructions. Quantity and quality of total RNA were determined by capillary electrophoresis on a RNA6000 bioanalyzer (Agilent Technologies). Two successive rounds of cDNA synthesis were applied to prepare 20ug of biotinylated target from one to two micrograms of total RNA (See Michaut et al., 2003).

source_name: Drosophila wandering larvae eye imaginal discs


title: WTlate03


title: Eye Discs iso4 lm17

description: To analyze gene expression in Drosophila eye primordia (wandering third instar larvae), the eye part was separated from the antennal part of the eye-antennal disc. One hundred eye imaginal discs were dissected and immediately transferred into 800ul Trizol (GIBCO Life Technologies)  and stored at –70°C until RNA extraction, which was performed according to the manufacturer’s instructions. Quantity and quality of total RNA were determined by capillary electrophoresis on a RNA6000 bioanalyzer (Agilent Technologies). Two successive rounds of cDNA synthesis were applied to prepare 20ug of biotinylated target from one to two micrograms of total RNA (See Michaut et al., 2003).


title: SGlate09


title: SGlate07


title: wild type 4 hours exposure to congo red 1 (cell wall chip)

status: Public on Mar 01 2005

city: Madrid

first: Javier

last: Arroyo

state: Madrid

address: Plaza Ramon y Cajal S/N

department: Microbiologia II

country: Spain

institute: Facultad de Farmacia (UCM)

description: S. cerevisiae was grown on YEPD. For Congo Red experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with Congo Red to a final concentration of 30 μg/ml. Cells were collected at 4 hours of growth, frozen at -80 °C and processed for RNA extraction.

organism: Saccharomyces cerevisiae

source_name: BY4741 Strain (Control mRNA: no treatment)

source_name: BY4741 Strain (4 hours Congo Red treatment)