title: P0239 dexamethasone pre-treatment

description: human myeloma cell  P0239 dexamethasone pre-treatment


title: P0270 dexamethasone pre-treatment

status: Public on Aug 04 2008

type: RNA

city: Little Rock

first: Yiming

last: Zhou

state: AR

address: 4301 West Markham St., Slot 776

country: USA

institute: Myeloma Institute for Research and Therapy

description: human myeloma cell  P0270 dexamethasone pre-treatment

hyb_protocol: Following fragmentation, 10 microg of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using GeneChip Scanner 3000

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method.

label: Biotin

molecule: total RNA

organism: Homo sapiens

label_protocol: Biotinylated cRNA were prepared according to the standard affymetrix protocol

extract_protocol: RNeasy Mini Kit(50)


title: P0251 dexamethasone pre-treatment

description: human myeloma cell  P0251 dexamethasone pre-treatment


title: P0084 thalidomide pre-treatment

description: human myeloma cell  P0084 thalidomide pre-treatment


title: P0387 dexamethasone pre-treatment

description: human myeloma cell  P0387 dexamethasone pre-treatment


title: P0255 thalidomide pre-treatment

description: human myeloma cell  P0255 thalidomide pre-treatment


title: P0099 dexamethasone pre-treatment

description: human myeloma cell  P0099 dexamethasone pre-treatment


title: P0237 thalidomide pre-treatment

description: human myeloma cell  P0237 thalidomide pre-treatment


title: P0075 dexamethasone pre-treatment

description: human myeloma cell  P0075 dexamethasone pre-treatment


title: P0175 dexamethasone pre-treatment

description: human myeloma cell  P0175 dexamethasone pre-treatment


title: P0202 thalidomide pre-treatment

description: human myeloma cell  P0202 thalidomide pre-treatment


title: YR_52hpf_rep3 (YR52_8-3)

city: West Lafayette

first: Yuk Fai

last: Leung

state: IN

address: 915 W. State Street

department: Biological Sciences

institute: Purdue University

description: Young mutant retina, 52hpf, pool of ten tissues, biological replicate 3

hyb_protocol: Hybridization, washing and staining were done according to a standard procedure from Affymetrix.

scan_protocol: Scanning was done according to a standard procedure from Affymetrix.

data_processing: The probe-level data were background adjusted, normalized and summarized by a dChip (http://www.dchip.org) algorithm using default parameters.

organism: Danio rerio

label_protocol: Thirty-three nanograms of total RNA were labeled by Affymetrix GeneChip Two-Cycle Target Labeling protocol.

growth_protocol: Fish and embryos were maintained according to Westerfield, M. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th edition. University of Oregon Press, Eugene, 2000.

extract_protocol: Total RNA was extracted from the pooled samples according to: Leung YF, Dowling JE. Gene Expression Profiling of Zebrafish Embryonic Retina. Zebrafish 2005:2:269-283.

treatment_protocol: Ten tissues were dissected from developing embryos according to: Leung YF, Dowling JE. Gene Expression Profiling of Zebrafish Embryonic Retina. Zebrafish 2005:2:269-283., and pooled as one biological replicate. The young mutants were staged by staging their wild-type siblings.

tag: Strain

value: AB

tag: Genotype

value: young

tag: Age

tag: Tissue

value: Retina


title: YR_36hpf_rep2 (YR36_7-2)

description: Young mutant retina, 36hpf, pool of twenty tissues, biological replicate 2

treatment_protocol: Twenty tissues were dissected from developing embryos according to: Leung YF, Dowling JE. Gene Expression Profiling of Zebrafish Embryonic Retina. Zebrafish 2005:2:269-283., and pooled as one biological replicate. The young mutants were staged by staging their wild-type siblings. The total number of individual embryos used was 10, which was the same as the other conditions. The only difference is that in this condition, both retinas were collected from the same embryo, as opposed to only one retina being collected in the other conditions. Nonetheless, the genetic variability was maintained because the same number of individual embryos was used.


title: YR_36hpf_rep3 (YR36_7-3)

description: Young mutant retina, 36hpf, pool of twenty tissues, biological replicate 3


title: YR_52hpf_rep1 (YR52_8-1)

description: Young mutant retina, 52hpf, pool of ten tissues, biological replicate 1


title: YA_36hpf_rep2 (YA36_5-2)

description: Young mutant whole embryos, 36hpf, pool of ten embryos, biological replicate 2

source_name: Young mutant whole embryo, 36hpf, pool of ten embryos

treatment_protocol: Ten embryos were collected according to: Leung YF, Dowling JE. Gene Expression Profiling of Zebrafish Embryonic Retina. Zebrafish 2005:2:269-283., and pooled as one biological replicate. The young mutants were staged by staging their wild-type siblings.

value: Whole embryo


title: YA_36hpf_rep3 (YA36_5-3)

description: Young mutant whole embryos, 36hpf, pool of ten embryos, biological replicate 3


title: YR_36hpf_rep1 (YR36_7-1)

description: Young mutant retina, 36hpf, pool of twenty tissues, biological replicate 1


title: YR_52hpf_rep2 (YR52_8-2)

description: Young mutant retina, 52hpf, pool of ten tissues, biological replicate 2


title: YA_52hpf_rep1 (YA52_6-1)

description: Young mutant whole embryos, 52hpf, pool of ten embryos, biological replicate 1

source_name: Young mutant whole embryo, 52hpf, pool of ten embryos


title: YA_52hpf_rep3 (YA52_6-3)

description: Young mutant whole embryos, 52hpf, pool of ten embryos, biological replicate 3


title: WR_36hpf_rep1 (WR36_3-1)

description: Wild type retina, 36hpf, pool of ten tissues, biological replicate 1

treatment_protocol: Ten tissues were dissected from developing embryos according to: Leung YF, Dowling JE. Gene Expression Profiling of Zebrafish Embryonic Retina. Zebrafish 2005:2:269-283., and pooled as one biological replicate.

value: Wild-type


title: WR_52hpf_rep1 (WR52_4-1)

description: Wild type retina, 52hpf, pool of ten tissues, biological replicate 1


title: WA_52hpf_rep2 (WA52_2-2)

description: Wild type whole embryos, 52hpf, pool of ten embryos, biological replicate 2

source_name: Wild type whole embryo, 52hpf, pool of ten embryos

treatment_protocol: Ten embryos were collected according to: Leung YF, Dowling JE. Gene Expression Profiling of Zebrafish Embryonic Retina. Zebrafish 2005:2:269-283., and pooled as one biological replicate.


title: WA_52hpf_rep3 (WA52_2-3-2)

description: Wild type whole embryos, 52hpf, pool of ten embryos, biological replicate 3


title: YA_52hpf_rep2 (YA52_6-2)

description: Young mutant whole embryos, 52hpf, pool of ten embryos, biological replicate 2


title: WR_36hpf_rep2 (WR36_3-2)

description: Wild type retina, 36hpf, pool of ten tissues, biological replicate 2


title: YA_36hpf_rep1 (YA36_5-1)

description: Young mutant whole embryos, 36hpf, pool of ten embryos, biological replicate 1


title: WA_36hpf_rep2 (WA36_1-2)

description: Wild type whole embryos, 36hpf, pool of ten embryos, biological replicate 2

source_name: Wild type whole embryo, 36hpf, pool of ten embryos


title: WR_52hpf_rep2 (WR52_4-2)

description: Wild type retina, 52hpf, pool of ten tissues, biological replicate 2


title: WR_36hpf_rep3 (WR36_3-3)

description: Wild type retina, 36hpf, pool of ten tissues, biological replicate 3


title: WA_36hpf_rep1 (WA36_1-1)

description: Wild type whole embryos, 36hpf, pool of ten embryos, biological replicate 1


title: WR_52hpf_rep3 (WR52_4-3)

description: Wild type retina, 52hpf, pool of ten tissues, biological replicate 3


title: Py-4-1_pcDNA_2

city: Zurich

first: Jay

middle: Woo

last: Shin

state: Zurich

address: Wolfgang-Paulistrasse 10; HCI H394

department: Institute of Pharmaceutical Sciences

country: Switzerland

institute: ETH Zurich

description: control-transfected Py-4-1 cell clone

hyb_protocol: Standard Affymetrix procedures were followed for hybridization and scanning of the arrays.

data_processing: Standard Affymetrix analysis using the MAS5 method.

label: biotin

organism: Mus musculus

source_name: Mouse hemangioendothelioma (Py-4-1 cell line)

label_protocol: Approximately 10 micrograms of total RNA was processed to produce biotinylated cRNA targets.

extract_protocol: Isolate total cellular RNA with 1 ml of the Trizol reagent (Invitrogen) after washing one 80-90 % confluent 10 cm culture dish with PBS. Homogenize the sample using a cell scraper and add 0.2 ml of chloroform in a 1.5 ml tube. Shake the sample vigorously for 15 seconds and allow standing for 5 minutes. Centrifuge the resulting mixture at 12,000 x g for 15 minutes at 4 degree Celsius. Transfer the aqueous phase to a fresh tube and add 0.5 ml of iso-propanol and mix. Allow the sample to stand for 5 minutes at room temperature and centrifuge at 12,000 x g for 10 minutes at 4 degrees Celsius. Remove the supernatant and wash the RNA pellet by adding 1 ml of 75% ethanol. Vortex the sample and then centrifuge at 12,000 x g for 5 minutes at 4 degrees Celsius. Remove the ethanol and briefly dry the RNA pellet for 5 minutes and add 50 micro-liter of RNAase/DNAase free water and mix by repeated pipetting until RNA pellet is fully dissolved.

tag: Py-4-1, a hemangioma derived cell line established from a Py-4 mouse cell line carrying polyoma virus early region, transfected with a control pcDNA vector.


title: Py-4-1_pcDNA/Prox1_1

description: stable Prox1-expressing Py-4-1 cell clone

scan_protocol: Standard Affymetrix procedures were followed for hybridization and scanning of the arrays.

tag: Py-4-1, a hemangioma derived cell line established from a Py-4 transgenic mouse line carrying polyoma virus early region, transfected with a pcDNA/Prox1 vector.


title: WA_36hpf_rep3 (WA36_1-3)

description: Wild type whole embryos, 36hpf, pool of ten embryos, biological replicate 3


title: 13489245 - PAC_8_2/2_2 vs PPR_8_5/2_1

status: Public on Aug 01 2008

city: Evry

first: Frederique

last: Bitton

address: 2 rue Gaston Cremieux

country: France

institute: INRA

description: -To understand how/if fertility and the mitochondrial background are interlinked. -To understand how the mitochondrial background influences the nuclear gene expression. -Compare and describe the total nuclear gene expression of CMS vs. fertile and CMS vs. Restored. -Describe and analyse genes that differ in expression (of special interest are genes that differs in both comparisons). -Group genes e.g. floral genes, highly expressed genes, transcription factors, nuclear encoded genes targeted for the mitochondrion. -Comparing two CMS-systems to elucidate differences and similarities between them.

hyb_protocol: PPR_8_5/2_1 Cy5 / PAC_8_2/2_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

scan_protocol: GenePix Pro 6.0, Cy3:pmt voltage 532nm,500V,laser power 30%, Cy5:635nm,pmt voltage 500V,laser power 30%

data_processing: The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.

organism: Brassica napus

label_protocol: labelling Cy3 and Cy5 indirect, amplification=yes, DNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).

growth_protocol: flowers - Plants were grown in a soil:perlite mixture under the following conditions: 16h photoperiod (light intensity 400umol/m2s); 85% air humidity; 22:C day/18:C night temperatures

treatment_protocol: no treatment

tag: Brassica napus (restord line rfo ppr)  - dev.stage (Boyes et al. Plant Cell 2001):boyes

value: between 6.00 et 6.50

tag: Brassica napus (pactol)  - dev.stage (Boyes et al. Plant Cell 2001):boyes


title: WA_52hpf_rep1 (WA52_2-1)

description: Wild type whole embryos, 52hpf, pool of ten embryos, biological replicate 1


title: Py-4-1_pcDNA/Prox1_2

tag: Py-4-1, a hemangioma derived cell line established from a Py-4 transgenic mouse cell line carrying polyoma virus early region, transfected with a pcDNA/Prox1 vector.


title: 13489246 - PPR_8_5/2_1 vs PAC_8_2/2_2

hyb_protocol: PAC_8_2/2_2 Cy5 / PPR_8_5/2_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Py-4-1_pcDNA_1

description: control-transfected Py-4-1 cell clone


title: 13489242 - PAC_8_2/2_2 vs CMS_8_4/2_2

hyb_protocol: CMS_8_4/2_2 Cy5 / PAC_8_2/2_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

tag: Brassica napus (cms-line)  - dev.stage (Boyes et al. Plant Cell 2001):boyes


title: 13489243 - PPR_8_5/2_1 vs CMS_8_4/2_2

hyb_protocol: CMS_8_4/2_2 Cy5 / PPR_8_5/2_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13489240 - CMS_8_4/2_2 vs PAC_8_2/2_2

hyb_protocol: PAC_8_2/2_2 Cy5 / CMS_8_4/2_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13372759 - 46_8_22/9_18/11_RNA vs 4:19_8_14/11_3

hyb_protocol: 4:19_8_14/11_3 Cy5 / 46_8_22/9_18/11_RNA Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

tag: Brassica napus (restored line 46)  - dev.stage (Boyes et al. Plant Cell 2001):boyes


title: 13372760 - 4:19_8_14/11_3 vs 46_8_22/9_18/11_RNA

hyb_protocol: 46_8_22/9_18/11_RNA Cy5 / 4:19_8_14/11_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13373288 - 4:19_0-5_9/2_1 vs 46_0-5_8/2_1

hyb_protocol: 46_0-5_8/2_1 Cy5 / 4:19_0-5_9/2_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13488972 - PAC_8_2/2_3 vs PPR_8_5/2_3

hyb_protocol: PPR_8_5/2_3 Cy5 / PAC_8_2/2_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13488973 - PPR_8_5/2_3 vs PAC_8_2/2_3

hyb_protocol: PAC_8_2/2_3 Cy5 / PPR_8_5/2_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13373285 - 46_0-5_8/2_1 vs 4:19_0-5_9/2_1

hyb_protocol: 4:19_0-5_9/2_1 Cy5 / 46_0-5_8/2_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13372758 - Ha_8_12/11_2 vs 4:19_8_14/11_3

hyb_protocol: 4:19_8_14/11_3 Cy5 / Ha_8_12/11_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

tag: Brassica napus (cv hanna)  - dev.stage (Boyes et al. Plant Cell 2001):boyes


title: 13488969 - PAC_8_2/2_3 vs CMS_8_4/2_3

hyb_protocol: CMS_8_4/2_3 Cy5 / PAC_8_2/2_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13489244 - CMS_8_4/2_2 vs PPR_8_5/2_1

hyb_protocol: PPR_8_5/2_1 Cy5 / CMS_8_4/2_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13488970 - PPR_8_5/2_3 vs CMS_8_4/2_3

hyb_protocol: CMS_8_4/2_3 Cy5 / PPR_8_5/2_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13488971 - CMS_8_4/2_3 vs PPR_8_5/2_3

hyb_protocol: PPR_8_5/2_3 Cy5 / CMS_8_4/2_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13488976 - Ha_0-5_13/11_1 vs Ha+3_0-5_27/1_1

hyb_protocol: Ha+3_0-5_27/1_1 Cy5 / Ha_0-5_13/11_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

tag: Brassica napus (cv hanna + a. thaliana chr 3)  - dev.stage (Boyes et al. Plant Cell 2001):boyes


title: 13373277 - Ha+3_0-5_27/1_1 vs Ha_0-5_13/11_1

hyb_protocol: Ha_0-5_13/11_1 Cy5 / Ha+3_0-5_27/1_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13372739 - Ha+3_8_31/1_2 vs Ha_8_12/11_2

hyb_protocol: Ha_8_12/11_2 Cy5 / Ha+3_8_31/1_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13373282 - Ha_0-5_13/11_1 vs 4:19_0-5_9/2_1

hyb_protocol: 4:19_0-5_9/2_1 Cy5 / Ha_0-5_13/11_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13488968 - CMS_8_4/2_3 vs PAC_8_2/2_3

hyb_protocol: PAC_8_2/2_3 Cy5 / CMS_8_4/2_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13372736 - Ha+3_8_31/1_2 vs 46_8_22/9_18/11_RNA

hyb_protocol: 46_8_22/9_18/11_RNA Cy5 / Ha+3_8_31/1_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13368672 - Ha_0-5_12/11_2 vs 4:19_0-5_14/11_3

hyb_protocol: 4:19_0-5_14/11_3 Cy5 / Ha_0-5_12/11_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365826 - 4:19_0-5_14/11_3 vs Ha_0-5_12/11_2

hyb_protocol: Ha_0-5_12/11_2 Cy5 / 4:19_0-5_14/11_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13372738 - Ha_8_12/11_2 vs Ha+3_8_31/1_2

hyb_protocol: Ha+3_8_31/1_2 Cy5 / Ha_8_12/11_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13372735 - 4:19_0-5_9/2_1 vs Ha_0-5_13/11_1

hyb_protocol: Ha_0-5_13/11_1 Cy5 / 4:19_0-5_9/2_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365822 - Ha+3_0-5_31/1_2 vs Ha_0-5_12/11_2

hyb_protocol: Ha_0-5_12/11_2 Cy5 / Ha+3_0-5_31/1_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13372737 - 46_8_22/9_18/11_RNA vs Ha+3_8_31/1_2

hyb_protocol: Ha+3_8_31/1_2 Cy5 / 46_8_22/9_18/11_RNA Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365825 - 46_0-5_8/2_1 vs Ha+3_0-5_27/1_1

hyb_protocol: Ha+3_0-5_27/1_1 Cy5 / 46_0-5_8/2_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13372757 - 4:19_8_14/11_3 vs Ha_8_12/11_2

hyb_protocol: Ha_8_12/11_2 Cy5 / 4:19_8_14/11_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365820 - 46_0-5_8/2_2 vs Ha+3_0-5_31/1_2

hyb_protocol: Ha+3_0-5_31/1_2 Cy5 / 46_0-5_8/2_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365824 - Ha+3_0-5_27/1_1 vs 46_0-5_8/2_1

hyb_protocol: 46_0-5_8/2_1 Cy5 / Ha+3_0-5_27/1_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365821 - Ha_0-5_12/11_2 vs Ha+3_0-5_31/1_2

hyb_protocol: Ha+3_0-5_31/1_2 Cy5 / Ha_0-5_12/11_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365819 - Ha+3_0-5_31/1_2 vs 46_0-5_8/2_2

hyb_protocol: 46_0-5_8/2_2 Cy5 / Ha+3_0-5_31/1_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365818 - 4:19_0-5_14/11_3 vs 46_0-5_8/2_2

hyb_protocol: 46_0-5_8/2_2 Cy5 / 4:19_0-5_14/11_3 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365817 - 46_0-5_8/2_2 vs 4:19_0-5_14/11_3

hyb_protocol: 4:19_0-5_14/11_3 Cy5 / 46_0-5_8/2_2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365812 - Ha_8_13/11_1 vs Ha+3_8_27/1_1

hyb_protocol: Ha+3_8_27/1_1 Cy5 / Ha_8_13/11_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365804 - 46_8_22/9_6/2_RNA vs 4:19_8_9/2_1

hyb_protocol: 4:19_8_9/2_1 Cy5 / 46_8_22/9_6/2_RNA Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365803 - Ha_8_13/11_1 vs 4:19_8_9/2_1

hyb_protocol: 4:19_8_9/2_1 Cy5 / Ha_8_13/11_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365802 - 4:19_8_9/2_1 vs Ha_8_13/11_1

hyb_protocol: Ha_8_13/11_1 Cy5 / 4:19_8_9/2_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365810 - Ha+3_8_27/1_1 vs 46_8_22/9_6/2_RNA

hyb_protocol: 46_8_22/9_6/2_RNA Cy5 / Ha+3_8_27/1_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13365811 - 46_8_22/9_6/2_RNA vs Ha+3_8_27/1_1

hyb_protocol: Ha+3_8_27/1_1 Cy5 / 46_8_22/9_6/2_RNA Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: IsletCellsCD40L_vs_IsletCellsCTRL_Exp3

city: Miami

first: Ricardo

last: Pastori

state: FL

department: Diabetes Research Institute

institute: University of Miami

description: the resulting images were analyzed with the software package GenePix Pro 5.1 (Axon Instruments at Molecular Devices). Data extracted from the images were transferred to the software package Acuity 4.0 (Axon Instruments) for normalization and statistical analysis. Each array was normalized for signal intensities across the whole array and locally, using Lowess normalization. Features for further analysis were selected according to the following quality criteria: (1) at least 90% of the pixels in the spot had intensity higher than background plus two standard deviations; (2), there were less than 2% saturated pixels in the spot; (3) signal to noise ratio (defined as ratio of the background subtracted mean pixel intensity to standard deviation of background) was 3 or above for each channel; (4) the spot diameter was between 80 and 110 micron; (5) the regression coefficient of ratios of pixel intensity was 0.6 or above.

hyb_protocol: Equal amounts of labeled aRNA were hybridized to Agilent Whole Human Genome Oligo microarrays (Agilent, Cat #G4112A) for 17 hours at 60oC according to manufacturer’s instructions

scan_protocol: The microarrays were scanned at 10 micron resolution using a GenePix 4000A scanner (Axon Instruments at Molecular Devices)

source_name: Depleted islet cells treated  5 hours with CD40L

label_protocol: Total RNA was subjected to two rounds of amplification using Amino Allyl MessageAmp aRNA Amplification Kit (Ambion, Cat#1753) and labeled with Cy5-NHS ethers (Amersham, Cat PA25001).

growth_protocol: Negative fraction (pure islet cells) were cultured over night in six well plate (9X105 cells per well/ ml) and treated with recombinant soluble human CD40L (100ng/ml plus 1ug/ml enhancer) for 5 hrs.

extract_protocol:
Total RNA was purified using PicoPure Kit (Arcturus), according to the manufacturer’s protocol, and quantified using the Nanodrop Spectrophotometer.
The RNA was then qualified on an Agilent 2100 Bioanalyzer.

treatment_protocol: Pancreatic islets were isolated and purified. Cultured for 24 hrs. at 37°C with 5% CO2 . Islets were dissociated 10 min with Acutase and depleted of Ductal cells using ductal marker antibody CA19-9 and anti mouse IgG magnetic beads.

tag: Sample

value: HP1696 Source:University of Miami DRI cGMP Gender:Male Age:33 years old

source_name: Depleted islet cells untreated

label_protocol: Total RNA was subjected to two rounds of amplification using Amino Allyl MessageAmp aRNA Amplification Kit (Ambion, Cat#1753) and labeled with Cy3- ethers (Amersham, Cat # PA23001).

growth_protocol: Negative fraction (pure islet cells) were cultured over night (9X105 cells per well/ ml)


title: 13365813 - Ha+3_8_27/1_1 vs Ha_8_13/11_1

hyb_protocol: Ha_8_13/11_1 Cy5 / Ha+3_8_27/1_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: IsletCellsCD40L_vs_IsletCellsCTRL_Exp1

data_processing: Data extracted from the images were transferred to the software package Acuity 4.0 (Axon Instruments) for normalization and statistical analysis. Each array was normalized for signal intensities across the whole array and locally, using Lowess normalization.

value: .HP1652 Source:University of Miami DRI cGMP   Gender: Female Age:53 years old


title: Hikone Male_LM2Dros405M2

city: Cambridge

first: Dean

last: Baker

address: Department of Genetics, Downing Street

country: United Kingdom

institute: University of Cambridge

description: Gene expression data from adult males.

hyb_protocol: Following fragmentation, cRNA were hybridized on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing: The data were normalized with the [R] Bioconductor Suite GC-RMA.

organism: Drosophila melanogaster

source_name: adult males

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Flies were reared and aged at 25C 0-6hrs post-eclosion

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

value: Japan Oregon R (Hikone)

tag: Gender

value: Male


title: 13372788 - 4:19_8_9/2_1 vs 46_8_22/9_6/2_RNA

hyb_protocol: 46_8_22/9_6/2_RNA Cy5 / 4:19_8_9/2_1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Hikone Female_LM2Dros407F1

description: Gene expression data from adult females.

source_name: adult females

value: Female


title: IsletCellsCTRL_vs_IsletCellsCD40L_Exp3

growth_protocol: Negative fraction (pure islet cells) were cultured over night in six well plate (9X105 cells per well/ ml).

growth_protocol: Negative fraction (pure islet cells) were cultured over night (9X105 cells per well/ ml) and treated with recombinant soluble human CD40L (100ng/ml plus 1ug/ml enhancer) for 5 hrs


title: IsletCellsCTRL_vs_IsletCellsCD40L_Exp1

value: .HP1652; Source:University of Miami DRI cGMP   Gender: Female Age:53 years old

growth_protocol: Negative fraction (pure islet cells) were cultured over night (9X105 cells per well/ ml) and treated with recombinant soluble human CD40L (100ng/ml plus 1ug/ml enhancer) for 5 hrs.


title: Hikone Male_LM2Dros404M1

description: Gene expression data from adult males.


title: Cambridge Male_LM2Dros423M2

description: Gene expression data from adult males.

value: Cambridge Oregon R


title: Cambridge Female_LM2Dros425F1


title: Hikone Male_LM2Dros406M3

description: Gene expression data from adult males.


title: Hikone Female_LM2Dros408F2

growth_protocol: Flies were reared 25C 0-6hrs post-eclosion


title: Cambridge Female_LM2Dros426F2


title: Cambridge Male_LM2Dros422M1

description: Gene expression data from adult males.


title: Cambridge Female_LM2Dros427F3


title: IsletCellsCD40L_vs_IsletCellsCTRL_Exp2

value: Hu 579 Source:Islet Center  City of Hope  California  Gender:Female  Age:49 years old


title: Cambridge Male_LM2Dros424M3

description: Gene expression data from adult males.


title: Antigua Male_LM2Dros411M2

description: Gene expression data from adult males.

value: Antigua


title: Glasgow Male_LM2Dros416M1

description: Gene expression data from adult males.

value: Glasgow Oregon R


title: Glasgow Male_LM2Dros418M3

description: Gene expression data from adult males.


title: Glasgow Male_LM2Dros417M2

description: Gene expression data from adult males.


title: Glasgow Female_LM2Dros420F2


title: Antigua Male_LM2Dros412M3

description: Gene expression data from adult males.


title: IsletCellsCTRL_vs_IsletCellsCD40L_Exp2


title: Antigua Female_LM2Dros414F2


title: Antigua Female_LM2Dros415F3


title: Glasgow Female_LM2Dros421F3


title: Antigua Female_LM2Dros413F1


title: Glasgow Female_LM2Dros419F1


title: France Male_LM2Dros399M2

description: Gene expression data from adult males.

value: France


title: France Male_LM2Dros400M3

description: Gene expression data from adult males.


title: Antigua Male_LM2Dros410M1

description: Gene expression data from adult males.


title: France Female_LM2Dros401F1


title: France Female_LM2Dros402F2


title: CG9238_males_2

city: Raleigh

first: Mike

last: Magwire

state: NC

address: 3501 Gardner Hall

department: Genetics

institute: NCSU

description: Seven lines homozygous for a single P-element insertion led to an increase in life span. Two replicates of each sex and line were used to identify differences in expression from the isogenic control line. Flies were aged 6 weeks (42 days) and frozen at approximately the same time (10:30 -12:00 AM) for RNA extraction.

hyb_protocol: Following fragmentation, 10 mkg of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

label: Biotinylated cRNA

source_name: whole bodies of 42 day old CG9238 male flies, replicate 2

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's (Affymetrix) instructions


title: CG9238_males_1

source_name: whole bodies of 42 day old CG9238 male flies, replicate 1


title: Asp mutated k-ras transfection_rep4

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

country: Germany

institute: Helmholtz Zentrum München GmbH

description: RNA isolation, Labelling, Hybridisation and Scanning procedure are described in the fields above

hyb_protocol: This hybridization mixture was placed on a prehybridised microarray, under a cover slip, placed into a hybridization chamber (Genetix) and immersed in a thermostatic bath at 42°C for at least 16 hours. After hybridization slides were washed in 40ml of 3x , 1x, 0.25x and 0.1x SSC at room temperature. For drying slides were placed in an empty 50 ml Falcon tube (Becton Dickinson, USA) and centrifuged at 4000m/s2. Beckers, J., Herrmann, F., Rieger, S., Drobyshev, A., Horsch, M., Hrabé de Angelis, M. and Seliger, B. (2005): Identification and validation of novel ERBB2 (Her2, NEU) targets including genes involved in angiogenesis. Int. J. Cancer 114: 590-597. Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schädler S, Erfle V, Beckers J, Schätzl H and Leib-Mösch C (2005): Cell Line Dependent RNA Expression Profiles of Prion-infected Mouse Neuronal Cells. JMB 349: 487-500 Seltmann M, Horsch M, Drobyshev A, Chen Y, Hrabé de Angelis M, Beckers J (2005): Assessment of a Systematic Expression Profiling. Approach in ENU-Induced Mouse Mutant Lines. Mam Genome 16 (1): 1-10

scan_protocol: Dried slides were scanned with a GenePix 4000A microarray scanner and the images were analyzed using the GenePix Pro3.0 image processing software (Axon Instruments, USA) Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schädler S, Erfle V, Beckers J, Schätzl H and Leib-Mösch C (2005): Cell Line Dependent RNA Expression Profiles of Prion-infected Mouse Neuronal Cells. JMB 349: 487-500 Seltmann M, Horsch M, Drobyshev A, Chen Y, Hrabé de Angelis M, Beckers J (2005): Assessment of a Systematic Expression Profiling. Approach in ENU-Induced Mouse Mutant Lines. Mam Genome 16 (1): 1-10

data_processing: Normalisation with GenePix Pro 6.0: Ratio based normalized data in each image so that the mean of ratio of means of all of the features is equal to 1. The VALUE column contains the log2 ratio of the result file after normalisation

source_name: Asp mutated k-ras transfected

label_protocol:
For labeling 20 µg of total RNA were used for reverse transcription and indirectly labeled with Cy3 or Cy5 fluorescent dye according the TIGR protocol (Hedge et al., 2000). Labeled cDNA was dissolved in 30 µl hybridization buffer (6x SSC, 0.5% SDS, 5x Denhardt’s solution and 50% formamide) and mixed with 30 µl of reference cDNA solution (pool from six control animals) labeled with the second dye.
HegdeP, Qi R, Abernathy R, Gay C, Dharap S, et al (2000): A concise guide to cDNA microarray analysis-II. Biotechniques 29: 548-562

extract_protocol: Total RNA was isolated just before processing for expression profiling. For preparation of total RNA individual organs were thawed in buffer containing chaotropic salt (RLT buffer, Qiagen) and homogenised using a Polytron homogeniser. Total RNA from individual samples was obtained according to manufacturer’s protocols using RNeasy Midi kits (Qiagen). The concentration was calculated from OD260/280 measurement and 2 µg RNA aliquots were run on a formaldehyde agarose gel to check for RNA integrity. The RNA was stored at -80°C in RNase free water (Qiagen).

tag: Asp mutated k-ras transfected NIH3T3 cell line

source_name: NIH3T3 (not transfected)

tag: NIH3T3 not transfected ES cell line


title: Asp mutated k-ras transfection_rep2


title: France Female_LM2Dros403F3

growth_protocol: Flies were reared and aged at 25C until 3-5 days post-eclosion.


title: Wt k-ras transfection_rep4

source_name: Wt k-ras transfected

tag: Wt k-ras transfected NIH3T3 cell line


title: France Male_LM2Dros398M1

description: Gene expression data from adult males.

tag: Genotype


title: Asp mutated k-ras transfection_rep1


title: Asp mutated k-ras transfection_rep3


title: Val mutated k-ras transfection_rep3

source_name: Val mutated k-ras transfected

tag: Val mutated k-ras transfected NIH3T3 cell line


title: Val mutated k-ras transfection_rep4


title: Mock k-ras transfection_rep4

source_name: Mock k-ras transfected

tag: Mock k-ras transfected NIH3T3 cell line


title: Val mutated k-ras transfection_rep2


title: Wt k-ras transfection_rep2


title: Mock k-ras transfection_rep3


title: Wt k-ras transfection_rep3


title: Mock k-ras transfection_rep2


title: Wt k-ras transfection_rep1


title: Mock k-ras transfection_rep1


title: timepoint 8 hrs t=0Cy3 - PBSCy5

city: Lelystad

first: Gabriele

last: Gross

address: P.O. Box 65

country: Netherlands

institute: Animal Sciences Group of Wageningen UR

description: slide 10: t=0-124-fluorescein/Cy3 vs PBS8-biotin/Cy5; dye-swap: slide 11

hyb_protocol: according to manufacturer's recommendations: RNA MICROMAX TSA labeling and detection kit (Perkin-Elmer, Zoetermeer, The Netherlands)

scan_protocol: scanned on Packard Bioscience BioChip Technologies apparatus (ScanArray Express, Perkin-Elmer) image processed with ScanArray Express software (Perkin-Elmer)

data_processing: data processing in GenePix Pro 5.0 (Molecular Devices, Apeldoorn, The Netherlands), data normalization (blank-specific background correction, LOWESS fit function with a fraction of 0.2) using a customized version of the statistical software package R, simultaneous data analysis of dye-swaps

label: Cy3 (fluorescein)

organism: Sus scrofa

source_name: Pooled total RNA of scrapings from samples taken at the beginning of the experiment (t=0)

label_protocol: 1 µg of RNA template (pooled) was labeled with fluorescein or biotin, RNA MICROMAX TSA labeling and detection kit (Perkin-Elmer, Zoetermeer, The Netherlands)

extract_protocol: Trizol extraction + additional steps to remove proteoglycan and polysaccharide contaminations + DNAse treatment

treatment_protocol: SISP test: pig jejunal segments, sampling of scrapings at t=0

tag: SISP test

tag: jejunum

tag: no perfusion

tag: samples taken at beginning of experiment

label: Cy5 (biotin)

source_name: Pooled total RNA of scrapings from jejunal segments perfused with PBS (control) for 8 hours

tag: 8 hours perfusion

tag: control

value: PBS

value: pig jejunal segments perfused with PBS (control), sampling of scrapings after 8 hours


title: timepoint 8 hrs PBSCy3 - t=0Cy5

description: slide 11: PBS8-fluorescein/Cy3 vs t=0-124-biotin/Cy5; dye-swap: slide 10

treatment_protocol: SISP test: pig jejunal segments perfused with PBS (control), sampling of scrapings after 8 hours

value: pig jejunal segments, sampling of scrapings at t=0


title: Val mutated k-ras transfection_rep1


title: H1299 cell infected with an p53 construct

city: Wuerzburg

first: Christof

last: Burek

address: Josef-schneider-Str. 2

department: Pathologie

institute: Universitaet Wuerzburg

description: standard Affymetrix

hyb_protocol: standard Affymetrix

scan_protocol: standard Affymetrix

data_processing: standard Affymetrix

source_name: H1299 cell line

label_protocol: standard Affymetrix

growth_protocol: standard Affymetrix

extract_protocol: RNeasy Kit (Qiagen)

treatment_protocol: standard Affymetrix


title: timepoint 8 hrs msakoCy3 - PBSCy5

description: slide 18: 8 hours; msako-fluorescein/Cy3 vs PBS-biotin/Cy5; dye-swap: slide 13

source_name: Pooled total RNA of scrapings from jejunal segments perfused with Lactobacillus plantarum 299v msa mutant suspension for 8 hours

treatment_protocol: SISP test: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v msa mutant), sampling of scrapings after 8 hours

tag: bacterial strain used

value: L. plantarum 299v msa mutant


title: timepoint 8 hrs PBSCy3 - SalmCy5

description: slide 9: 8 hours; PBS-fluorescein/Cy3 vs Salm-biotin/Cy5; dye-swap: slide 8

source_name: Pooled total RNA of scrapings from jejunal segments perfused with Salmonella typhimurium suspension for 8 hours

value: S. typhimurium

value: pig jejunal segments perfused with bacterial suspensions (S. typhimurium), sampling of scrapings after 8 hours


title: timepoint 8 hrs SalmCy3 - PBSCy5

description: slide 8: 8 hours; Salm-fluorescein/Cy3 vs PBS-biotin/Cy5; dye-swap: slide 9

treatment_protocol: SISP test: pig jejunal segments perfused with bacterial suspensions (S. typhimurium), sampling of scrapings after 8 hours


title: timepoint 8 hrs PBSCy3 - msakoCy5

description: slide 13: 8 hours; PBS-fluorescein/Cy3 vs msako-biotin/Cy5; dye-swap: slide 18

value: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v msa mutant), sampling of scrapings after 8 hours


title: 18S z000509 KLF4-2 CY5 vs STD-2 CY3

city: St Lucia

first: Darrin

middle: F

last: Taylor

state: Qld

address: Research Rd

department: Genomics and Bioinformatics

country: Australia

institute: Institute for Molecular Bioscience, Uni of Qld

description: Pairwise comparison of STD-MO vs klf4-MO

hyb_protocol: Followed protocol described in: PMID: 15998744

data_processing: Imagene5.6, Lowess Normalisation (R)

source_name: klf4 MO

label_protocol: Indirect aRNA Ambion

extract_protocol: RNAAmplification Ambion

tag: Total RNA from pooled 18S Fertilized embryos from natural matings injected with a klf4 specific antisense morpholino oligonucleotide (klf4-MO)

source_name: STD MO

tag: Total RNA from pooled 18S Fertilized embryos from natural matings injected with a standard nonsense morpholino oligonucleotide (Std-MO)


title: timepoint 4 hrs srtkoCy3 - PBSCy5

description: slide 21-2: 4 hours; srtko-fluorescein/Cy3 vs PBS-biotin/Cy5; dye-swap: slide 22

source_name: Pooled total RNA of scrapings from jejunal segments perfused with Lactobacillus plantarum 299v srt mutant suspension for 4 hours

treatment_protocol: SISP test: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v srt mutant), sampling of scrapings after 4 hours

tag: 4 hours perfusion

value: L. plantarum 299v srt mutant

source_name: Pooled total RNA of scrapings from jejunal segments perfused with PBS (control) for 4 hours

value: pig jejunal segments perfused with PBS (control), sampling of scrapings after 4 hours


title: H1299 Cell line infected with an truncated p73 adenovirus

description: standard Affymetrix


title: 18S z000511 KLF4-4 CY5 vs STD-4 CY3

scan_protocol: Followed protocol described in: PMID: 15998744


title: timepoint 8 hrs PBSCy3 - srtkoCy5

description: slide 15: 8 hours; PBS-fluorescein/Cy3 vs srtko-biotin/Cy5; dye-swap: slide 23-2

source_name: Pooled total RNA of scrapings from jejunal segments perfused with Lactobacillus plantarum 299v srt mutant suspension for 8 hours

value: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v srt mutant), sampling of scrapings after 8 hours


title: H1299 Cell line infected with an truncated p53 adenovirus

description: standard Affymetrix

source_name: h1299 cell line


title: timepoint 4 hrs PBSCy3 - msakoCy5

description: slide 17: 4 hours; PBS-fluorescein/Cy3 vs msako-biotin/Cy5; dye-swap: slide 16

treatment_protocol: SISP test: pig jejunal segments perfused with PBS (control), sampling of scrapings after 4 hours

source_name: Pooled total RNA of scrapings from jejunal segments perfused with Lactobacillus plantarum 299v msa mutant suspension for 4 hours

value: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v msa mutant), sampling of scrapings after 4 hours


title: 18S z000508 STD-1 CY5 vs KLF4-1 CY3


title: timepoint 8 hrs PBSCy3 - WTCy5

description: slide 7: 8 hours; PBS-fluorescein/Cy3 vs WT-biotin/Cy5; dye-swap: slide 6-2

source_name: Pooled total RNA of scrapings from jejunal segments perfused with wildtype Lactobacillus plantarum 299v suspension for 8 hours

value: L. plantarum 299v wildtype

value: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v wildtype), sampling of scrapings after 8 hours


title: H1299 cell infected with an GFP construct

description: standard Affymetrix

source_name: Human Cell Line H1299


title: H1299 cell line infected with p73 containing adenoviral construct

description: standard Affymetrix

source_name: H1299 cell lline

tag: standard Affymetrix


title: timepoint 4 hrs WTCy3 - PBSCy5

description: slide 4: 4 hours; WT-fluorescein/Cy3 vs PBS-biotin/Cy5; dye-swap: slide 5

source_name: Pooled total RNA of scrapings from jejunal segments perfused with wildtype Lactobacillus plantarum 299v suspension for 4 hours

treatment_protocol: SISP test: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v wildtype), sampling of scrapings after 4 hours


title: 18S z000510 STD-1 CY5 vs KLF4-1 CY3


title: timepoint 4 hrs PBSCy3 - srtkoCy5

description: slide 22: 4 hours; PBS-fluorescein/Cy3 vs srtko-biotin/Cy5; dye-swap: slide 21-2

value: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v srt mutant), sampling of scrapings after 4 hours


title: timepoint 4 hrs msakoCy3 - PBSCy5

description: slide 16: 4 hours; msako-fluorescein/Cy3 vs PBS-biotin/Cy5; dye-swap: slide 17

treatment_protocol: SISP test: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v msa mutant), sampling of scrapings after 4 hours


title: timepoint 4 hrs PBSCy3 - WTCy5

description: slide 5: 4 hours; PBS-fluorescein/Cy3 vs WT-biotin/Cy5; dye-swap: slide 4

value: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v wildtype), sampling of scrapings after 4 hours


title: timepoint 8 hrs WTCy3 - PBSCy5

description: slide 6-2: 8 hours; WT-fluorescein/Cy3 vs PBS-biotin/Cy5; dye-swap: slide 7

treatment_protocol: SISP test: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v wildtype), sampling of scrapings after 8 hours


title: C40 vs. pLRT - repeat 4 - mAdbID:51262

city: Bethesda

first: Kathleen

last: Kelly

state: MD

address: 37 Convent Drive

department: Cancer and Cell Biology Branch

institute: National Institutes of Health

description: mAdb experiment ID: 51262

hyb_protocol: NCI cDNA Microarray Hybridization Other: For pre-hybrization, apply 40 ul of pre-hybridization buffer (5X SSC, 0.1% SDS, 1% BSA) to the array and incubate at 42°C for at least 30 minutes and up to an hour. Wash off the pre-hybridization solution by rapidly plunging the slide in distilled water for 2 minutes, then transfer slide to 100% isopropanol for 2 minutes. Allow slide to air dry completely prior to use. (Can spin dry if in a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying before setting up hybridization.) For hybridization, combine Cy3 and Cy5 labeled targets together (~9 ul recovered for each). Add 1 µl COT-1 DNA (8-10 ug/ul) and 1 ul poly A (8-10 ug/ul). Denature target at 100°C for 1 minute, then snap cool on ice. (Final volume should be about 20 ul.) Make fresh 2X Formamide hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) and warm to 42°C just before adding to samples. Add 20 ul of 2X F-hyb buffer to samples. Load 40 ul sample onto microarray. Add 20 ul of 3X SSC to wells in hyb chamber to maintain humidity. Incubate overnight (12-16 hours) at 42°C in water bath or hybridization oven. After hybridization of slides, wash slides for 2 minute in 2X SSC with 0.1% SDS (with occasional plunging), for 2 minute in 1X SSC (with occasional plunging), for 2 minutes in 0.2X SSC (with occasional plunging), for 1 minute in 0.05X SSC, and spin for 3 minutes at 650 rpm to dry. (Refer to "NCI Microarray Manual")

scan_protocol: Creator: GenePix Pro 4.0.0.54 Scanner: GenePix 4000A [47890] ScanPower: 100;; 100;; n/a;; n/a LaserPower: 1.25;; 1.91;; n/a;; n/a Temperature: 36.56

data_processing:
Data Processing Protocol
Calculation Method: Signal intensities of microarray features were calculated by subtracting the median local background from the median signal intensity. Features were considered for analysis if in the range 50-300ìm with signal intensity greater than 100 in both channels that were greater than one standard deviation above background with at least a 2:1 signal to background ratio. Signal intensities for an entire microarray were normalized to the 50% percentile median value, to correct for superior cy3 or cy5 labeling of one sample versus the other, or to superior cDNA synthesis of one sample versus the other. After filtering and normalization, the cy3 and cy5 values for the pLRT control cDNA and the experimental (V12 or effector domain) cDNA were expressed as a ratio to indicate the fold up- or down-regulation relative to the pLRT control cDNA.

source_name: pLRT - Cy5

label_protocol:
Cy5 Labeling Protocol
Other: cDNA was labeled with Cy5 monofunctional reactive dye to amino allyl-modified dUTP incorporated into cDNA using the FairPlay Microarray labeling kit (Stratagene, La Jolla, CA).

extract_protocol:
Trizol Extraction
Extraction method: guanidium HCL/phenol-chloroform
Other: Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA).

treatment_protocol:
Treatment type: compound
Agent: Doxycycline
Treatment dose: 250 ng/ml
Treatment time: 72 hours

tag: Cell line

value: HME16C human mammary epithelial cells

tag: Cell type

value: hTERT immortalized human mammary epithelial cells

tag: Other

value: These cells were infected with the control pLRT retroviral expression vector that does not express a gene product.

label_protocol:
Cy3 Labeling Protocol
Other: cDNA was labeled with Cy3 monofunctional reactive dye to amino allyl-modified dUTP incorporated into cDNA using the FairPlay Microarray labeling kit (Stratagene, La Jolla, CA).

value: These cells were infected with the pLRT-H-RasV12C40 retroviral expression vector that directs the doxycycline-inducible expression of activated RasV12C40.

tag: genotype/variation


title: C40 vs. pLRT - repeat 2 - mAdbID:51260

description: mAdb experiment ID: 51260

scan_protocol: Creator: GenePix Pro 4.0.0.54 Scanner: GenePix 4000A [47890] ScanPower: 100;; 100;; n/a;; n/a LaserPower: 1.36;; 1.92;; n/a;; n/a Temperature: 35.75


title: timepoint 8 hrs srtkoCy3 - PBSCy5

description: slide 23-2: 8 hours; srtko-fluorescein/Cy3 vs PBS-biotin/Cy5; dye-swap: slide 15

treatment_protocol: SISP test: pig jejunal segments perfused with bacterial suspensions (L. plantarum 299v srt mutant), sampling of scrapings after 8 hours

value: PBS

value: pig jejunal segments perfused with PBS (control), sampling of scrapings after 8 hours


title: C40 vs. pLRT - repeat 3 - mAdbID:51261

description: mAdb experiment ID: 51261

scan_protocol: Creator: GenePix Pro 4.0.0.54 Scanner: GenePix 4000A [47890] ScanPower: 100;; 100;; n/a;; n/a LaserPower: 1.32;; 1.91;; n/a;; n/a Temperature: 36.14

source_name: pLRT - Cy3


title: C40 vs. pLRT - repeat 1 - mAdbID:51259

description: mAdb experiment ID: 51259

scan_protocol: Creator: GenePix Pro 4.0.0.54 Scanner: GenePix 4000A [47890] ScanPower: 100;; 100;; n/a;; n/a LaserPower: 1.24;; 1.93;; n/a;; n/a Temperature: 35.43