title: Exon_Erythroid_Day14_01

city: Santa Clara

first: Jason

middle: Wray

last: Hubbard

address: 3380 Central Expressway

institute: Affymetrix Inc

description: CD34-positive early hematopoietic progenitors isolated from growth factor mobilized peripheral blood; The culture media contained 15% FCS, 15% human AB serum, Iscove’s modified Dulbecco’s medium (IMDM), 10ng/ml IL-3, 2 units ml Epo, and 50 ng/ml SCF. SCF was gradually decreased, and IL-3 was not added after day 3. Cells were collected at day 14.

hyb_protocol: Hybridized for 16 hours at 45deg according to the Affymetrix WT Sense Target Labeling Assay Manual; Arrays were washed and stained on an Affymetrix FS450 Fluidics Station using the Affymetrix Hybridization, Wash, and Stain Kit according to the directions in the Affymetrix WT Sense Target Labeling Assay Manual.

scan_protocol: Arrays were scanned on an Affymetrix GCS3000 Scanner

data_processing: The input files were normalized with full quantile normalization (Irizarry et al 2003).  For each input array, for each probe expression value, the array ith percentile probe value was replaced with the average of all array ith percentile points.  Next, the 5,359,576 probes were manipulated into the analysis values as follows.  Probes with GC count less than 6 and greater than 17 were excluded from the analysis.  Probe scores were then transformed by taking the Natural Logarithm of 0 plus the probe score.  Exon arrays do not use individual mis-match probes.  Background is established from a pool of probes designed for that purpose.  Background probes are stratified by CG content and are defined in the HumanExon10ST_antigenomic.bgp file.  BGP files can also be downloaded from www.affymetrix.com.  Each probe score was corrected for background by subtracting the median expression score of background probes with similar GC content.    Exon level expression patterns were analyzed using the XRAY software (Biotique Systems, Inc., version 2.5)

source_name: Hematopoietic progenitors 14 days after initiation of differentiation

label_protocol: Globin- and ribosomal RNA-depleted RNA was amplified and labeled according to the Affymetrix WT Sense Target Labeling Assay Manual

growth_protocol: An in vitro primary culture system was employed to generate cells at different stages of human erythroid differentiation16. The culture system used was CD34-positive early hematopoietic progenitors isolated from growth factor mobilized peripheral blood (purchased from ALL Cells, Berkeley, CA) to promote the erythroid differentiation program. The culture media contained 15% FCS, 15% human AB serum, Iscove’s modified Dulbecco’s medium (IMDM), 10ng/ml IL-3, 2 units ml Epo, and 50 ng/ml SCF.

extract_protocol: Total cellular RNA was extracted from each culture at early (day 7), intermediate (day 10), and late (day14) stages of differentiation. Alpha- and beta-globin mRNAs were depleted from the RNA samples using GLOBINclearTM Kit (Ambion, Inc.). 1 μg of total globin-depleted RNA was used to subtract ribosomal RNA using the RibominusTM Human/Mouse Transcriptome Isolation kit (Invitrogen).

treatment_protocol: SCF was gradually decreased, and IL-3 was not added after day 3. Cells were collected at day 7, 10, and 14, which respectively correspond to basophilic (early), polychromatic (intermediate), and orthochromatic (late) erythroblasts. 97-99% of these cells were erythroid as determined by flow  cytometry for glycophorin A and CD71.

tag: Day 14


title: 13688918 - Col0_ABA1 vs Col0_C1

status: Public on Jul 01 2009

type: RNA

city: BEAUCOUZE

first: Sandra

last: Pelletier

address: 42, rue Georges Morel - BP 60057

country: France

institute: INRA

description: Transcriptomic study of lpp2 KO mutant in response to abcsisic acid

hyb_protocol: Col0_C1 Cy5 / Col0_ABA1 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 3.0, Cy3:pmt voltage 532nm,500V,laser power 35%, 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.

molecule: total RNA

organism: Arabidopsis thaliana

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, aRNA 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: leaf - media : 2/3 soil 1/3 vermiculite Hygrometry : 60-80% temperature : 22°C light : 8h photoperiod

treatment_protocol: Name:Control - control buffer - control buffer,water+silwet:quantity 0.05percent time 2min . 2min and 1h of incubation, 2min and 2h of incubation

tag: Arabidopsis thaliana (col-0)  - age:6week

treatment_protocol: Name:ABA - abiotic stress - hormone,abiotic stress,aba:quantity 0.01mM time 1min . Soaking 1 min then incubation 3 hours


title: Exon_Erythroid_Day14_03

state: CA

country: USA

label: biotin

organism: Homo sapiens


title: Exon_Erythroid_Day14_02


title: 13688919 - Col0_C1 vs Col0_ABA1

hyb_protocol: Col0_ABA1 Cy5 / Col0_C1 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: 13689484 - lpp2_C2 vs lpp2_ABA2

hyb_protocol: lpp2_ABA2 Cy5 / lpp2_C2 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: Arabidopsis thaliana (col-0)  mutant (Atlpp2.2 -/-) - age:6week


title: Exon_Erythroid_Day10_03

description: CD34-positive early hematopoietic progenitors isolated from growth factor mobilized peripheral blood; The culture media contained 15% FCS, 15% human AB serum, Iscove’s modified Dulbecco’s medium (IMDM), 10ng/ml IL-3, 2 units ml Epo, and 50 ng/ml SCF. SCF was gradually decreased, and IL-3 was not added after day 3. Cells were collected at day 10.

source_name: Hematopoietic progenitors 10 days after initiation of differentiation

tag: Day 10


title: Exon_Erythroid_Day07_01

description: CD34-positive early hematopoietic progenitors isolated from growth factor mobilized peripheral blood; The culture media contained 15% FCS, 15% human AB serum, Iscove’s modified Dulbecco’s medium (IMDM), 10ng/ml IL-3, 2 units ml Epo, and 50 ng/ml SCF. SCF was gradually decreased, and IL-3 was not added after day 3. Cells were collected at day 7.

source_name: Hematopoietic progenitors 7 days after initiation of differentiation

tag: Day 7


title: Exon_Erythroid_Day10_02


title: Exon_Erythroid_Day07_02


title: 13688920 - lpp2_C1 vs lpp2_ABA1

hyb_protocol: lpp2_ABA1 Cy5 / lpp2_C1 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: BASE-A space experiment simulation microgravity

status: Public on Jun 30 2009

city: MOL

first: Natalie

last: Leys

state: Please select one below:

address: 200, boeretang

department: Institute for Environment, Health & Safety

country: Belgium

description: no additional information

hyb_protocol: The two differentially labeled cDNAs were resuspended in the universal hybridization buffer (Pronto kit; Corning-Promega, United States), mixed, and added jointly to the surface of the spotted microarray glass slide for overnight hybridization at 42°C using a HS 4800 Pro automated hybridization station (TECAN, Switzerland). The slide was washed according to the Pronto kit’s protocol and then dried in the hybridization station.

data_processing: Spots were analyzed using GenePix Pro v.6.0.1 software and flagged according to their quality. Quality check was performed for each array using the ArrayQuality package from BioConductor (Gentleman et al, 2004). Raw median intensity data were imported into R version 2.7.0 for statistical analysis using the LIMMA package version 2.14.1 (Smyth, 2005) as available from BioConductor. Raw data were background-corrected based on convolution of normal and exponential distributions with an offset of 50 (Ritchie et al, 2007). Data were normalized within each array using the loess normalization algorithm (Smyth and Speed, 2003) and between each array using Aquantile normalization (Yang and Thorne, 2003). The in-slide replicate correlations were calculated using the Duplicate correlation function in the LIMMA package (Smyth et al, 2005). The log expression values were fit to a linear model and moderated t-statistics were calculated using empirical Bayes method (Smyth, 2004). P-values were corrected for multiple testing using the Benjamin and Hochberg’s method to control the false discovery rate (Benjamini and Hochberg, 1995).

organism: Rhodospirillum rubrum

source_name: Rhodospirillum rubrum S1H grown during a 10 day ground control experiment

label_protocol: RNA amplification was performed using consecutively both MessageAmp™ II-Bacteria Kit for prokaryotic RNA amplification producing the amplified RNA (aRNA) and Amino Allyl MessageAmpTM II aRNA Amplification kit (Ambion, United States) producing the Amino Allyl RNA (aaRNA) following the manufacturer’s instructions. The quality/integrity of total RNA and amplified RNA was checked using a BioAnalyzer 2100 (Agilent, United States). aaRNA was labeled using the ChipShot™ Indirect Labeling and Clean-Up System (Promega and Corning, United States) following the manufacturer’s instructions. The ChipShot™ Indirect System provides reagents and enzymes for generating fluorescently labeled cDNA using a two-step labeling process that includes synthesis of aminoallyl-modified cDNA, followed by conjugation to the CyDye™ NHS ester.

extract_protocol: Total RNA was extracted using a SV Total RNA Isolation System kit (Promega, United States) and stored at -80 °C. The quality/integrity of total RNA was checked using a BioAnalyzer 2100 (Agilent, United States) and total RNA quantity was measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, United States).

tag: R. rubrum strain S1H was obtained from the American Type Culture Collection (ATCC25903). Sistrom-succinate medium (Sistrom, 1960) was supplemented with 2 % agar.

source_name: Rhodospirillum rubrum S1H grown during a 10 day simulation of space microgravity

growth_protocol: R. rubrum S1H cultures, prepared according to the space flight experimental setups described in BASE-A sample, were cultivated in the ESA random positioning machine (RPM) facility located at the University of Sassari (Italy) (Figure 3). The RPM was operated as a random walk 3D-clinostat (basic mode) with an angular velocity of rotation of 60 deg/s for 10 days.


title: 0.04 fmole of miRNAs

status: Public on Jul 30 2009

type: mixed

city: Minneapolis

first: Xiaoxiao

last: Zhang

address: 312 Church St. SE

institute: University of Minnesota

description: We hybridized different amounts of microRNAs from the mirVana miRNA Reference Panel v9.1 to microarrays.

hyb_protocol: A mixture of labeled RNA and DNA was hybridized to a miRNA microarray (Thomson et al) inside a MAUI mixer in a MAUI hybridization system (BioMicro® System Inc Salt Lake City UT USA).     Thomson J.M. Parker J. Perou C.M. and S.M. Hammond. 2004. A custom microarray platform for analysis of microRNA gene expression. Nat. Methods 1: 47-53. Epub 2004 Sep 29.

scan_protocol: Slides were scanned with a ScanArray 5000 machine (Perkin Elmer® Waltham MA USA).

data_processing: BlueFuse (BlueGenome Cambridge UK) was used to quantify pixel intensities.  GeneSpring GX 7.3.1 (Agilent Technologies) was used for data normalization.

organism: synthetic construct

source_name: miRNAs from mirVana miRNA Reference Panel v9.1

label_protocol: Different amounts of miRNA standards in the panel  were ligated to a synthetic linker 5’-pCU-DY547-3’.   DNA oligonucleotides (Invitrogen) complementary to a subset of mammalian miRNAs were combined and labeled with a ULYSIS® Alexa Fluor® 647 kit.

extract_protocol: commercially available

tag: synthetic RNAs coresponding to human miRNAs

label: Alexa Fluor® 647

molecule: other

source_name: synthetic

tag: synthetic DNA oligos corrresponding to human miRNAs


title: BASE-A space experiment simulation ionizing radiation

description: no additional information

scan_protocol: The two differentially labeled cDNAs were resuspended in the universal hybridization buffer (Pronto kit; Corning-Promega, United States), mixed, and added jointly to the surface of the spotted microarray glass slide for overnight hybridization at 42°C using a HS 4800 Pro automated hybridization station (TECAN, Switzerland). The slide was washed according to the Pronto kit’s protocol and then dried in the hybridization station.

source_name: Rhodospirillum rubrum S1H grown during a 10 day simulation of space ionizing radiation

growth_protocol: R. rubrum S1H agar cultures prepared according to the space flight experimental setups described in BASE-A sample were exposed to ionizing radiations in the calibration room facility at SCK•CEN (Mol, Belgium). In order to mimic the ISS ionizing radiation environment, pure 137Cs gamma and 252Cf neutron sources were used as representative of low-linear energy transfert (LET) and high-LET particles respectively. The source reference values were traceable to primary standards (PTB for gamma and NPL for neutrons). During irradiation, samples were kept rotating at 2 rpm in between the two sources for 10 days (maximum irradiation time allowance) at 21°C ± 2°C, being exposed to a total dose of ca. 2 mGy (Figure 3). Actual absorbed dose was assessed in situ by using TLD's (LiF:Mg,Ti 'TLD-100' from Harshaw Bicron, United Stated and LiF:Mg,Cu,P 'MCP-N' from TLD-Poland, Poland) and OSLD's (Al2O3:C 'TLD-500' from Harshaw-Bicron) for gamma detection and bubble detectors (BD-PND-BDT from Bubble Technology Industry, Canada) for neutron detection.


title: Exon_Erythroid_Day07_03


title: 13688921 - lpp2_ABA1 vs lpp2_C1

hyb_protocol: lpp2_C1 Cy5 / lpp2_ABA1 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: MESSAGE 2 space experiment

description: no additional information

label_protocol: Ten micrograms of RNA was reverse transcribed by random priming according to the Pronto kit’s instructions (Corning-Promega, United States) and directly labeled by incorporation of Cy3-dCTP or Cy5-dCTP nucleotides (Amersham Bioscience, United Kingdom) for respectively control and experimental samples.

tag: R. rubrum strain S1H was obtained from the American Type Culture Collection (ATCC25903). Sistrom-succinate medium (Sistrom, 1960) was supplemented with 3 % peptone, 1 % yeast extract (BD Biosciences, USA) and 2 % agar (Sistrom-peptone-yeast) (Saegesser et al, 1992).

source_name: Rhodospirillum rubrum S1H grown during a 10 day space flight in ISS

growth_protocol:
Three independent cultures of R. rubrum S1H grown to stationary phase in dark aerobic conditions were resuspended in 0.85 % NaCl (Saline tablets, BRS, Oxoid) to a final OD680 of 0.600 and transported at room temperature from SCK•CEN (Mol, Belgium) to Baikonour (Kazakhstan) 8 days before launch. Inoculation was performed 15 hours before launch on Sistrom-peptone-yeast plates. Drops of 106 cells of R. rubrum S1H, as well as dilution series for viable count purpose, were spotted in biological triplicates spread over 2 agar plates. After inoculation, Petri plates were first sealed with parafilm and Kapton tape next, individually packed in Ziplock bag and then jointly packed in a second Ziplock bag and placed in a sealed jar. Radiation dosimeters (Track-Etch Detectors TED's, Optically Stimulated Luminescence Detectors OSLD's and ThermoLuminescent Detectors TLD's, described in Goossens et al., 2006) and temperature sensors were added in the jar. This jar was then wrapped in protective foam and NOMEX fabric bags and stored at room temperature (Figure 1). Finally, the exterior of the package was disinfected with 3 % hydrogen peroxide wipes according to Russian flight procedures and was placed into the Soyuz (TMA-3) 8 hours before launch.
Upon arrival in the ISS (after 2 days of flight in the Soyuz), the containers were incubated for 8 days in the Russian Service Module of the ISS behind structural bars (Figure 1), at a relative constant temperature of 21 °C ± 2 °C. After a total of 10 days flight (October, 18-28 2003) in the ISS, the samples returned back to Earth (Soyuz TMA-2).
After landing in the Kazakhstan desert, the jar containing the cultures in Petri dishes was transferred to an active cooling ground transportation box at 4 °C and transported without exposure to the airport X-rays scanning. The container arrived in SCK•CEN (Mol, Belgium), 40 hours after the return of the Soyuz vehicle.


title: BASE-A space experiment

description: no additional information

tag: R. rubrum strain S1H was obtained from the American Type Culture Collection (ATCC25903). Sistrom-succinate medium (Sistrom, 1960) was supplemented with  2 % agar.

growth_protocol:
Cultures of R. rubrum S1H grown as mentioned above were transported at room temperature from SCK•CEN (Mol, Belgium) to Baikonour (Kazakhstan) 10 days before launch. Three biological independent culture suspensions were deposited 24 hours before launch as 4 spots of 20 µl corresponding to 6x106 cells or as 1 mat of 100 µl corresponding to 3x107 cells per ca. 10 cm² surface of a 5 ml layer Sistrom-succinate agar medium in 6 well culture plates (CellStar 6, Greiner Bio-One, Belgium) and kept at room temperature.
An oxygen indicator strip (Anaerotest, Merck) was added at the bottom of the multiwell plate in between the wells, to visualise oxygen concentration in the gas phase in the multiwell plate during incubation pre-, in- and post-flight. Culture plates were sealed with 1 layer of Parafilm® and 1 layer of Scotch® tape. Radiation dosimeters (TED's and OSLD's described in Vanhavere et al., 2008) and temperature sensors were added in the container.  Two culture plates were sealed hermetically in 1 polycarbonate 'Biocontainer' (PedeoTechniek, Belgium) (described in Vanhavere et al, 2008) and vacuum sealed in a highly transparent Minigrip®polyethylene bag of 60 µm thick. During the 2-days trip in the Soyuz TMA-9 to the ISS, the temperature of the pouch was maintained at 21-23 °C. After docking, all samples were stored at ambient temperature in the ISS Russian Zvezda service module (the exact location is unknown) and a temperature of 19-22 °C was recorded. The samples returned back to Earth after a total of 12 days in-flight (September, 17-28 2006).


title: Treatment 2: AA30 (EHEC 86-24 pH 5.0, followed by 30 min pH 3.0)

status: Public on Jun 26 2009

city: Toronto

first: Debora

last: Foster

state: Ontario

address: 350 Victoria Street

department: Chemistry and Biology

country: Canada

institute: Ryerson University

description: Before normalization, each individual array spot was pruned to exclude bad or empty spots flagged by GenePix Pro 3.0. Normalization was preformed using the LOWESS algorithm. For each point, three biological and two technical replicates were measured, resulting in a maximum of 6 gene expression values per gene per treatment.

hyb_protocol: Eighty-one micro-litres of prewarmed AdvaHybC™ hybridisation solution (Advalytix, Germany) was added to each sample. The samples were then denatured at 95oC for 3 minutes then cooled to 42oC. The samples were loaded onto the microarrays and incubated in the SlideBooster™ (Advalytix, Germany) at 42oC overnight. After hybridisation, the microarray was washed 3 times for 10 to 15 minutes in 1× SSC, 0.1% sodium dodecyl sulfate solution at 50°C after removal of the coverslip in room-temperature 1× SSC. The slide was rinsed in room temperature 1× SSC, then 0.1× SSC. The slide was dried at 25×g for 5 minutes.

scan_protocol: Microarrays were scanned using the Agilent G2565BA scanner; this yielded two 16-bit TIFF images per array, one corresponding to the Cy3 channel and one corresponding to the Cy5 channel. These images were then quantified using GenePix Pro 3.0 (Molecular Devices, USA).

data_processing: Signal intensities were averaged among the technical replicates. Two types of data analysis were performed: to indentify density-dependent changes in gene expression, single averaged normalized signal intensities for each treatment point were compared to the average of corresponding unstressed control signal intensities. Genes which showed a relative signal log2ratio (SLR) value above 1.0 or below -1.0 were selected for further analysis.

organism: Escherichia coli

source_name: Cy5 labeled EHEC acid-adapted in DMEM pH 5, followed by 30 minutes pH 3

label_protocol: 20 micrograms total RNA was reverse-transcribed with the use of SuperScript II RNase H- Reverse Transcriptase and random primers (Invitrogen, USA, 150 ng/µl final concentration) in the presence of 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dATP, 0.15 mM dTTP (GE Healthcare, USA), and 0.15 mM of aminoallyl-dUTP (Sigma, USA). The reaction was allowed to proceed for two hours at 42°C. To stop the reaction and hydrolyse the remaining RNA sodium hydroxide was added to a final concentration of 160 mM and the samples were heated at 65 oC for 15 minutes. The samples were neutralised and the cDNA was purified using the CyScribe™ GFX purification kit (GE Healthcare, USA). Aminoallyl labelled cDNA then underwent a chemical reaction with Cy3 or Cy5 monofunctional NHS-Esters (GE Healthcare, USA). This reaction was stopped by the addition of hydroxylamine to a final concentration of 1.24 M. Labelled samples were then purified using the CyScribe™ GFX purification kit (GE Healthcare, USA). Purified labelled cDNA was dried in a speed vac, just until dry, and then resuspended to a final volume of 9 µl including 45 µg of calf thymus DNA (Sigma, USA) and 45 µg of yeast tRNA (Invitrogen, USA).

growth_protocol: Bacteria were grown in LB broth overnight and subcultured in DMEM prior to acid stress

extract_protocol: RNA was extracted using Trizol (Invitrogen)

treatment_protocol: Acid adaptation was done in DMEM at pH 5.0 at room temperature, followed by acid-stress at pH 3.0 for 30 minutes

tag: EHEC strain 86-24

source_name: Cy3 labeled EHEC grown in DMEM pH 7.4

treatment_protocol: Control treatment. Bacteria were grown in DMEM pH 7.4 (unstressed)


title: Treatment 1: UA30 (EHEC 86-24 exposed to pH 3 for 30 min)

source_name: Cy5 labeled EHEC acid-stressed in DMEM pH 3, 30minutes

treatment_protocol: Acid shocking was done in DMEM at pH 3.0 at room temperature for 30 minutes

source_name: Cy3 labeled EHEC grown in DMEM pH 7.4, 15 minutes

growth_protocol: Bacteria were grown in LB broth overnight and subcultured in DMEM


title: MESSAGE 2 space experiment simulation ionizing radiation

description: no additional information

tag: R. rubrum strain S1H was obtained from the American Type Culture Collection (ATCC25903). Sistrom-succinate medium (Sistrom, 1960) was supplemented with 3 % peptone, 1 % yeast extract (BD Biosciences, USA) and 2 % agar  (Sistrom-peptone-yeast) (Saegesser et al, 1992).

growth_protocol: R. rubrum S1H agar cultures prepared according to the space flight experimental setups described in MESSAGE 2 sample were exposed to ionizing radiations in the calibration room facility at SCK•CEN (Mol, Belgium). In order to mimic the ISS ionizing radiation environment, pure 137Cs gamma and 252Cf neutron sources were used as representative of low-linear energy transfert (LET) and high-LET particles respectively. The source reference values were traceable to primary standards (PTB for gamma and NPL for neutrons). During irradiation, samples were kept rotating at 2 rpm in between the two sources for 10 days (maximum irradiation time allowance) at 21°C ± 2°C, being exposed to a total dose of ca. 2 mGy (Figure 3). Actual absorbed dose was assessed in situ by using TLD's (LiF:Mg,Ti 'TLD-100' from Harshaw Bicron, United Stated and LiF:Mg,Cu,P 'MCP-N' from TLD-Poland, Poland) and OSLD's (Al2O3:C 'TLD-500' from Harshaw-Bicron) for gamma detection and bubble detectors (BD-PND-BDT from Bubble Technology Industry, Canada) for neutron detection.


title: MESSAGE 2 space experiment simulation microgravity

description: no additional information

growth_protocol: R. rubrum S1H cultures, prepared according to the space flight experimental setups described in MESSAGE 2 sample, were cultivated in the ESA random positioning machine (RPM) facility located at the University of Sassari (Italy) (Figure 3). The RPM was operated as a random walk 3D-clinostat (basic mode) with an angular velocity of rotation of 60 deg/s for 10 days.


title: HJAY_Erythroid_Day7_05

data_processing: .CEL files were processed in Expression Console using RMA for probeset summarization.


title: Normal stroma, slide 9540, sample 1

status: Public on Jun 01 2009

city: Houston

first: Chad

last: Creighton

state: TX

address: One Baylor Plaza, Mail Stop: BCM305

department: Biostatistics, Ducan Cancer Center

institute: Baylor College of Medicine

description: human normal peripheral zone prostate stroma, compared to universal reference RNA

hyb_protocol: The sample and reference probes were mixed with GEx hybridization buffer and blocking solution (Agilent, Santa Clara, CA) and denatured by a 2-minute incubation at 95 degC. Probes were hybridized to Agilent 4x44K Whole Human Genome arrays and rotating over night at 65 degC.

scan_protocol: Slide were scanned in an Agilent Microarray Scanner and the data were analyzed using Agilent Feature Extraction Software (v.9.1).

data_processing: LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent and Bioconductor software was used.

label: Alexa Fluor 647

source_name: normal prostate stroma

label_protocol: Complementary DNA (cDNA) reverse transcription and fluorescent labeling reactions were carried out using SuperScript Plus Direct cDNA Labeling system (Invitrogen, Carlsbad, CA).

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.  Microarray analysis was performed using 2.5 micrograms of total RNA.

tag: normal peripheral zone prostate stroma

label: Alexa Fluor 555

source_name: human, universal reference RNA

tag: Stratagene, Universal Human Reference RNA Catalog #740000


title: Normal stroma, slide 21023, sample 1


title: Normal stroma, slide 9540, sample 2


title: Normal stroma, slide 21022, sample 1


title: Treatment 3: UA15 (EHEC 86-24 exposed to pH 3 for 15 min)

description: Before normalization, each individual array spot was pruned to exclude bad or empty spots flagged by GenePix Pro 3.0. Normalization was preformed using the LOWESS algorithm.  For each point, three biological and two technical replicates were measured, resulting in a maximum of 6 gene expression values per gene per treatment.

data_processing: Signal intensities were averaged among the technical replicates.  Two types of data analysis were performed: to indentify density-dependent changes in gene expression, single averaged normalized signal intensities for each treatment point were compared to the average of corresponding unstressed control signal intensities.  Genes which showed a relative signal log2ratio (SLR) value above 1.0 or below -1.0 were selected for further analysis.

source_name: Cy5 labeled EHEC acid-stressed in DMEM pH 3, 15 minutes

treatment_protocol: Acid shocking was done in DMEM at pH 3.0 (unbuffered) at room temperature for 15 minutes


title: Normal stroma, slide 21020, sample 1


title: Normal stroma, slide 9538, sample 1


title: Normal stroma, slide 9538, sample 2


title: Normal stroma, slide 21022, sample 2


title: Normal stroma, slide 9539, sample 1


title: Normal stroma, slide 21021, sample 2


title: HJAY_Erythroid_Day7_04


title: Normal stroma, slide 21021, sample 1


title: HJAY_Erythroid_Day14_01


title: Normal stroma, slide 9541, sample 1


title: HJAY_Erythroid_Day14_02


title: Normal stroma, slide 21020, sample 2


title: HJAY_Erythroid_Day7_03


title: Normal stroma, slide 9539, sample 2


title: Normal stroma, slide 9541, sample 2


title: HJAY_Erythroid_Day7_01


title: HJAY_Erythroid_Day7_02


title: Normal stroma, slide 9537, sample 1


title: Cancer stroma, slide 21022, sample 2

description: human grade 3 reactive stroma prostate cancer tissues, compared to universal reference RNA

source_name: prostate cancer-associated stroma


title: HJAY_Erythroid_Day14_03

source_name: Hematopoietic progenitors 14 days after initiation of differentiation


title: Cancer stroma, slide 21021, sample 2

tag: grade 3 reactive stroma prostate cancer tissues


title: Cancer stroma, slide 21020, sample 1


title: Cancer stroma, slide 21021, sample 1


title: Cancer stroma, slide 9541, sample 1


title: Cancer stroma, slide 21023, sample 1


title: Cancer stroma, slide 21022, sample 1


title: Cancer stroma, slide 9541, sample 2


title: HJAY_Erythroid_Day14_05


title: Cancer stroma, slide 9538, sample 2


title: Normal stroma, slide 9537, sample 2


title: Cancer stroma, slide 9537, sample 2


title: Cancer stroma, slide 9539, sample 2


title: HepG2_Ag2CO3 rep2

city: Sapporo

first: Akinobu

last: Ito

address: North-13, West-8, Kitaku

department: Graduate School of Engineering

country: Japan

institute: Hokkaido University

description: HepG2_Ag2CO3 rep2

hyb_protocol: Target preparation and hybridization was performed according to One-Cycle Eukaryotic Target Labeling Assay protocols described in Affymetrix technical manual (Affymetrix, Santa Clara, CA). Labeled cRNA was fragmented by incubation at 94 °C for 35 min in the presence of 40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate. Ten ug of fragmented cRNA was hybridized to a Human Genome Focus array (Affymetrix, Santa Clara, CA) containing probes for 8795 human genes for 16 h at 45 °C. After hybridization, the microarrays were automatically washed and stained with streptavidin-phycoerythrin by using a fluidics station 450 (Affymetrix, Santa Clara, CA).

scan_protocol: probe arrays were scanned with the Genechip System confocal scanner (Affymetrix, Santa Clara, CA).

data_processing: The data were analyzed with MAS5 using Avadis 4.2 prophetic (Strand Genomics, Redwood City, CA).

source_name: Hep G2 treated with silver carbonate for 24h

label_protocol: Target preparation was performed according to One-Cycle Eukaryotic Target Labeling Assay protocols described in Affymetrix technical manual (Affymetrix, Santa Clara, CA). cDNA was synthesized from the 5 ug of total RNA by using One-Cycle cDNA Synthesis kit (Invitrogen, Carlsbad, CA) with a T7-(dT)24 primer incorporating a T7 RNA polymerase promoter. cRNA was synthesized from the cDNA and biotin-labeled by in vitro transcription using IVT Labeling kit (Affymetrix, Santa Clara, CA).

growth_protocol: Cells were cultured in Eagle's minimal essential medium (MEM) (Nissui, Tokyo, Japan) supplemented with 1% non-essential amino acid (Invitrogen, Carlsbad, CA), 10% fetal bovine serum and 60 mg/ml kanamycin at 37 °C and 5% CO2.

extract_protocol: The cells were were washed with phosphate buffered saline (PBS) and lysed directly on culture dishes. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany).

treatment_protocol: Cells were grown to 70% confluency in 60 mm culture dishes and exposed to 1.3 mg/l of silver carbonate for 24h at 37 °C.

tag: Cell bank no

tag: Cell name

value: Hep G2

tag: Sex

value: male

tag: Age of sampling

value: 15 years

tag: Tissue derived

value: liver

tag: Case history

value: hepatocyte carcinoma

tag: Life span

value: infinite

tag: Classification

value: Transformed


title: HepG2_Ag2CO3 rep3

description: HepG2_Ag2CO3 rep3


title: HepG2_nanoAg+cysteine rep2

description: HepG2_nanoAg+cysteine rep2

source_name: Hep G2 treated with silver nanoparticles and cysteine for 24h

treatment_protocol: Cells were grown to 70% confluency in 60 mm culture dishes and exposed to 1 mg/l of silver nanoparticles and 5mM of N-acetyl-L-cysteinefor 24h at 37 °C.


title: HepG2_nanoAg+cysteine rep3

description: HepG2_nanoAg+cysteine rep3


title: HepG2_nanoAg+cysteine rep1

description: HepG2_nanoAg+cysteine rep1


title: Cancer stroma, slide 9540, sample 2


title: Cancer stroma, slide 9538, sample 1


title: Cancer stroma, slide 9540, sample 1


title: Cancer stroma, slide 9539, sample 1


title: Cancer stroma, slide 9537, sample 1


title: HepG2_PS rep3

description: HepG2_PS rep3

source_name: Hep G2 treated with polystyrene nanoparticles for 24h

treatment_protocol: Cells were grown to 70% confluency in 60 mm culture dishes and exposed to 1 mg/l of polystyrene nanoparticles (15 nm) for 24h at 37 °C.


title: HepG2_PS rep2

description: HepG2_PS rep2


title: HepG2_nanoAg rep2

description: HepG2_nanoAg rep2

source_name: Hep G2 treated with silver nanoparticles for 48h

treatment_protocol: Cells were grown to 70% confluency in 60 mm culture dishes and exposed to 1 mg/l of silver nanoparticles (7-10 nm) for 24h at 37 °C.


title: HepG2_nanoAg rep3

description: HepG2_nanoAg rep3

source_name: Hep G2 treated with silver nanoparticles for 24h


title: Disruption of B. thetaiotaomicron ECF-sigma factor BT1053 in MM-neutral O-glycans replicate 2

city: Ann Arbor

first: Eric

middle: Charles

last: Martens

state: MI

address: 1150 West Medical Center Drive

department: Dept. of Microbiology and Immunology

institute: University of Michigan Medical School

description: Bacteroides thetaiotaomicron VPI-5482; mutant in ECF-sigma factor BT1053; grown in minimal medium (MM) plus neutral O-glycans as sole carbon source

hyb_protocol: Affymetrix

scan_protocol: Affymetrix

data_processing: GeneChip intensity values normalized to a value of 500 using Affymetrix Microarray Suite 5 software (MAS5)

organism: Bacteroides thetaiotaomicron VPI-5482

source_name: Bacteroides thetaiotaomicron VPI-5482 total RNA

label_protocol: Affymetrix

extract_protocol: Affymetrix


title: HepG2_PS rep1

description: HepG2_PS rep1


title: Cancer stroma, slide 21020, sample 2


title: Disruption of B. thetaiotaomicron anti-sigma factor BT3992 in MM-glucose replicate 1

description: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT3992; grown in minimal medium (MM) plus glucose as sole carbon source


title: Disruption of B. thetaiotaomicron anti-sigma factor BT4356 in MM-glucose replicate 3

description: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT4356; grown in minimal medium (MM) plus glucose as sole carbon source


title: HepG2_nanoAg rep1

description: HepG2_nanoAg rep1


title: Disruption of B. thetaiotaomicron SusC-like transporter BT1042 in MM-neutral O-glycans replicate 2

description: Bacteroides thetaiotaomicron VPI-5482; mutant in SusC-like transporter BT1042; grown in minimal medium (MM) plus neutral O-glycans as sole carbon source


title: HepG2_control rep4

description: HepG2_control rep4

source_name: human hepatoma cell line, Hep G2


title: Disruption of B. thetaiotaomicron SusC-like transporter BT1042 in MM-neutral O-glycans replicate 1

tag: Bacteroides thetaiotaomicron VPI-5482; mutant in SusC-like transporter BT1042; grown in minimal medium (MM) plus neutral O-glycans as sole carbon source


title: HepG2_control rep5

description: HepG2_control rep5


title: Disruption of B. thetaiotaomicron anti-sigma factor BT3992 in MM-glucose replicate 2

tag: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT3992; grown in minimal medium (MM) plus glucose as sole carbon source


title: Disruption of B. thetaiotaomicron ECF-sigma factor BT1053 in MM-neutral O-glycans replicate 1

tag: Bacteroides thetaiotaomicron VPI-5482; mutant in ECF-sigma factor BT1053; grown in minimal medium (MM) plus neutral O-glycans as sole carbon source


title: Disruption of B. thetaiotaomicron anti-sigma factor BT3992 in MM-glucose replicate 3


title: HepG2_control rep6

description: HepG2_control rep6


title: Disruption of B. thetaiotaomicron anti-sigma factor BT4356 in MM-glucose replicate 2

tag: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT4356; grown in minimal medium (MM) plus glucose as sole carbon source


title: Disruption of B. thetaiotaomicron anti-sigma factor BT4249 in MM-glucose replicate 3

description: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT4249; grown in minimal medium (MM) plus glucose as sole carbon source


title: Disruption of B. thetaiotaomicron anti-sigma factor BT4249 in MM-glucose replicate 2.1

tag: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT4249; grown in minimal medium (MM) plus glucose as sole carbon source


title: co-cultured pMBMEC - 1

city: Zurich

first: Francois

last: Verrey

address: Winterthurerstr. 190

department: Institute of Physiology

country: Switzerland

institute: University of Zurich

description: gene expression from 5 day cultures on transwell filters of mouse brain microvascular endothelial cells co-cultured with 3 week mouse brain glial cultures in the lower well in non-contact

hyb_protocol: The biotin-labelled cDNA targets were mixed in 220 µL of Hybridization Mix (Affymetrix Inc., P/N 9007200) containing Hybridization controls and Control Oligonucleotide B2 (Affymetrix Inc., P/N 900454). Samples were hybridized to GeneChip® arrays for 18 h at 45 ºC

scan_protocol: An Affymetrix GeneChip® Scanner 3000 (Affymetrix Inc.) was used to measure fluorescent intensities emitted by labelled targets and signals were saved to a CEL file format.

data_processing: CEL file date were normalized using RMAExpress 0.5 (Robust Multichip Average) (Bolstad et al 2003) and Custom CDF version 10 (Mm430_Mm_REFSEQ.cdf) (Dai et al 2005). The RMAExpress options background-adjustment and quantile normalization were enabled. To exclude probe sets in the lower end of the signal range which have large signal variation, a cutoff value was defined as the maximal signal value obtained from non-murine Affymetrix-control probe sets multiplied by a factor of 1.2. In this data set, the cutoff value was log25.78. Probe sets with a signal below cutoff in every array of the corresponding comparison, as well as Affymetrix control probe sets (prefix of “AFFX”) were excluded from further analysis.

organism: Mus musculus

source_name: co-cultured mouse brain endothelial cells

label_protocol: cDNAs were prepared by reverse transcription (RT) (WT-Ovation Pico System, NuGEN) according to the manufacturer’s protocol from total isolated RNAs. Fragmented and biotin labelled single-stranded cDNA targets were generated with the FL-Ovation cDNA Biotin Module V2 (NuGEN, 4200-12) according to the manufacturer’s protocol.

growth_protocol: mouse brain microvascular endothelial cells were isolated with or without culture as described above

extract_protocol: RNA was extracted using RNAready kit (BioDiagnostik, University Bern, Bern, Switzerland) according to the manufacturer protocol including an on-column DNase digestion

treatment_protocol: as above

tag: for each preparation cortices from ten 4 week old female C57BL/6 mice (Charles River, L’Arbresle, France)


title: co-cultured pMBMEC - 2


title: HepG2_Ag2CO3 rep1

description: HepG2_Ag2CO3 rep1


title: cell-line MBMEC - 2

description: gene expression from b.End5 endothelioma cultures

source_name: b.End5 cell-line

tag: b.End5 endothelioma cultures from mouse brain microvascular cells


title: Disruption of B. thetaiotaomicron anti-sigma factor BT4356 in MM-glucose replicate 1


title: co-cultured pMBMEC - 3


title: cell-line MBMEC - 1


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1618 in MM-glucose replicate 3

description: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT1618; grown in minimal medium (MM) plus glucose as sole carbon source


title: single-cultured pMBMEC - 3

description: gene expression from 5 day cultures on transwell filters of mouse brain microvascular endothelial cells

source_name: single-cultured mouse brain endothelial cells


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1052 complemented with BT1052 in MM-glucose replicate 1

description: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT1052 complemented with wild-type copy of BT1052; grown in minimal medium (MM) plus glucose as sole carbon source


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1618 in MM-glucose replicate 2

tag: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT1618; grown in minimal medium (MM) plus glucose as sole carbon source


title: cell-line MBMEC - 3


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1052 in MM-glucose replicate 3

description: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT1052; grown in minimal medium (MM) plus glucose as sole carbon source


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1052 complemented with BT1052 in MM-glucose replicate 2

tag: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT1052 complemented with wild-type copy of BT1052; grown in minimal medium (MM) plus glucose as sole carbon source


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1618 in MM-glucose replicate 1


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1052 in MM-glucose replicate 1

tag: Bacteroides thetaiotaomicron VPI-5482; mutant in anti-sigma factor BT1052; grown in minimal medium (MM) plus glucose as sole carbon source


title: single-cultured pMBMEC - 2


title: non-cultured ppMBMEC - 3

description: gene expression from highly pure mouse brain microvascular endothelial cells

source_name: non-cultured PECAM1+ mouse brain endothelial cells


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1052 complemented with BT1052 in MM-glucose replicate 3


title: non-cultured ppMBMEC - 2


title: Disruption of B. thetaiotaomicron anti-sigma factor BT1052 in MM-glucose replicate 2


title: single-cultured pMBMEC - 1


title: primary_fibroblasts_500cGy

status: Public on May 31 2009

city: Salt Lake City

first: Philip

middle: J

last: Moos

state: UT

department: Pharmacology & Toxicology

institute: University of Utah

description: primary fibroblasts irradiated at 500 cGy compared to unirradiated primary fibroblasts

hyb_protocol: Agilent two-color GE hyb/wash protocol

scan_protocol: Agilent 10 micron scanning protocol, Agilent Technologies Scanner G2505B US45102802

data_processing: Feature extraction and processing - Agilent FE 8.1.1.1 protocol, lowess normaliztion and background subtracted

source_name: primary fibroblasts - gamma irradation - 500 cGy, 4 hr time point

label_protocol: Agilent two-color LRILAK labeling protocol

growth_protocol: All cells were grown to quiescence; primary cells were grow to confluence and maintain for 1 week prior to irradiation and RNA collection, immortalized cells were grown to confluence and maintained for 2 days prior to irradiation and RNA collection.

extract_protocol: Total RNA was collected using Qiagen RNeasy Mini kits.

treatment_protocol: Cells were non-treated or irradiated with a 137Cs source at a dose rate of 4.7 cGy/min, for a total of 10, 100 or 500 cGy.

source_name: primary fibroblasts - untreated

tag: untreated


title: immortalized_fibroblasts_10cGy

description: hTert immortalized fibroblasts irradiated with 10 cGy compared to hTert immortalized fibroblasts

source_name: HCA2 fibroblasts - gamma irradation - 10 cGy, 4 hr time point

source_name: HCA2 fibroblasts - untreated


title: immortalized_fibroblasts_100cGy

description: hTert immortalized fibroblasts irradiated with 100 cGy compared to hTert immortalized fibroblasts

source_name: HCA2 fibroblasts - gamma irradation - 100 cGy, 4 hr time point


title: non-cultured ppMBMEC - 1


title: immortalized_fibroblasts_500cGy

description: hTert immortalized fibroblasts irradiated with 500 cGy compared to hTert immortalized fibroblasts

source_name: HCA2 fibroblasts - gamma irradation - 500 cGy, 4 hr time point


title: primary_fibroblasts_100cGy

description: primary fibroblasts irradiated at 100 cGy compared to unirradiated primary fibroblasts

source_name: primary fibroblasts - gamma irradation - 100 cGy, 4 hr time point


title: immortalized_keratinocytes_500cGy

description: hTert immortalized keratinocytes irradiated with 500 cGy compared to hTert immortalized keratinocytes

source_name: N-Tert1keratinocytes - gamma irradation - 500 cGy, 4 hr time point

source_name: N-Tert1keratinocytes - untreated


title: Disruption of B. thetaiotaomicron anti-sigma factor BT4249 in MM-glucose replicate 1


title: rat morphine neuron 3

status: Public on May 04 2009

description: microRNA expression and opioid signaling

hyb_protocol: A mixture of labeled RNA and DNA was hybridized to a miRNA microarray (Thomson et al), inside a MAUI mixer in a MAUI hybridization system (BioMicro® System, Inc, Salt Lake City, UT, USA).     Thomson, J.M., Parker, J., Perou, C.M., and S.M. Hammond. 2004. A custom microarray platform for analysis of microRNA gene expression. Nat. Methods 1: 47-53. Epub 2004 Sep 29.

scan_protocol: Slides were scanned with a ScanArray 5000 machine (Perkin Elmer®, Waltham, MA, USA).

data_processing: BlueFuse (BlueGenome, Cambridge, UK) was used to quantify pixel intensities.  GeneSpring GX 7.3.1 (Agilent Technologies) was used to normalize data with a percentile method.  We first analyzed data of a microarray experiment performed on the same day, and then compared results or conclusions from the experiment (typically one biological replicate) to those from others to identify consistent microRNA expression changes in different biological replicates.

organism: Rattus norvegicus

source_name: rat neuron, morphine treated

label_protocol: Total RNA was ligated to a synthetic linker, 5’-pCU-DY547-3’.  Reference DNA oligonucleotides (Invitrogen) complementary to a subset of mammalian miRNAs were combined and labeled with a ULYSIS® Alexa Fluor® 647 kit.

growth_protocol: Six to eight-week-old CD1(ICR) male mice was obtained from Charles River (Charles River Laboratories, Portage, MI) at least one week before experiment.  Primary cells were prepared from the dissected hippocampi of newborn rats and maintained in culture for three weeks before experiment.

extract_protocol: Trizol® reagent (Invitrogen) was used to extract total RNA from all the samples.

treatment_protocol: Nine mice were anesthetized intraperitoneally (90 mg/kg ketamine and 10 mg/kg xylazine) and implanted with micro-osmotic pump (Alzet Corp., Palo Alto, CA, 1ul/hr 72 hr). The mice were divided into three groups, infused with saline control, 12μg/hr morphine, or 0.31μg/hr fentanyl. After three days' infusion, mice were euthanized and the cerebellum and hippocampus regions dissected. Primary neuronal cultures were treated with 1μM morphine or 10nM fentanyl for three days.

tag: cells derived from tissues dissected from animals

molecule: genomic DNA

source_name: synthetic DNA oligos

tag: synthetic DNA oligos corrresponding to human miRNAs


title: primary_fibroblasts_10cGy

description: primary fibroblasts irradiated at 10 cGy compared to unirradiated primary fibroblasts

source_name: primary fibroblasts - gamma irradation - 10 cGy, 4 hr time point


title: primary_keratinocytes_100cGy

description: primary keratinocytes irradiated at 100 cGy compared to unirradiated primary keratinocytes

source_name: primary keratinocytes - gamma irradation - 100 cGy, 4 hr time point

source_name: primary keratinocytes - untreated


title: primary_keratinocytes_500cGy

description: primary keratinocytes irradiated at 500 cGy compared to unirradiated primary keratinocytes

source_name: primary keratinocytes - gamma irradation - 500 cGy, 4 hr time point


title: primary_keratinocytes_10cGy

description: primary keratinocytes irradiated at 10 cGy compared to unirradiated primary keratinocytes

source_name: primary keratinocytes - gamma irradation - 10 cGy, 4 hr time point


title: rat control neuron 3

source_name: rat neuron, control treated


title: rat morphine neuron 2


title: rat fentanyl neuron 2

source_name: rat neuron, fentanyl treated


title: immortalized_keratinocytes_10cGy

description: hTert immortalized keratinocytes irradiated with 10 cGy compared to hTert immortalized keratinocytes

source_name: N-Tert1keratinocytes - gamma irradation - 10 cGy, 4 hr time point


title: immortalized_keratinocytes_100cGy

description: hTert immortalized keratinocytes irradiated with 100 cGy compared to hTert immortalized keratinocytes

source_name: N-Tert1keratinocytes - gamma irradation - 100 cGy, 4 hr time point


title: rat morphine neuron 1


title: rat fentanyl neuron 1


title: mouse morphine hippocampus 2

source_name: mouse hippocampus, morphine treated

tag: tissues dissected from animals


title: mouse morphine hippocampus 1


title: mouse morphine hippocampus 3


title: rat control neuron 1


title: rat control neuron 2


title: rat fentanyl neuron 3


title: mouse fentanyl hippocampus 2

source_name: mouse hippocampus, fentanyl treated


title: mouse fentanyl hippocampus 1


title: mouse fentanyl hippocampus 3


title: mouse control hippocampus 2

source_name: mouse hippocampus, control treated


title: mouse morphine cerebellum 3

source_name: mouse cerebellum, morphine treated


title: mouse morphine cerebellum 1


title: mouse control cerebellum 1

source_name: mouse cerebellum, control treated


title: WT_+N_26_rep1 (set 2)

status: Public on Apr 30 2009

city: Uruma

first: Kenichi

last: Sajiki

address: 12-22 Suzaki

institute: OIST-IRP

description: Gene expression profile under nitrogen rich condition at 26ºC in fcp1-452 mutant strain of fission yeast.

hyb_protocol: Affymetrix standard protocol (overnight)

scan_protocol: Affymetrix standard protocol using Affymetirix Fluidics 450 and Scanner 3000

data_processing: Background correct with RMA by GeneSpring GX 7.3.1. Normalization was carried out as following three steps. (1) Data transformation: Sets values below 0.01 to the cutoff. (2) Per chip: Normalize each chip to the 75th percentile of the measuerments taken from that chip. (3) Per Gene: Normalize each gene to the median of the measuerments for that gene.

label: Biotin

molecule: polyA RNA

organism: Schizosaccharomyces pombe

source_name: L972, logarithmically growing in EMM2 at 26ºC

label_protocol: Affymetrix sample processing protocol

growth_protocol: t=26 or 37 degrees C, incubated in EMM2 liquid medium, harvested in log phase.

extract_protocol: Total RNA was isolated by acid phenol methods described in http://www.sanger.ac.uk/PostGenomics/S_pombe/, and then polyA-RNA was purified by Oligotex-dT30<super>mRNA purification kit (Takara).

tag: L972 h- wild type


title: mouse control hippocampus 3


title: mouse control cerebellum 2


title: mouse morphine cerebellum 2


title: WT_+N_26_rep2 (set 2)


title: mouse fentanyl cerebellum 1

source_name: mouse cerebellum, fentanyl treated


title: WT_+N_37_rep1 (set 2)

source_name: L972, logarithmically growing in EMM2 at 37ºC


title: mouse control hippocampus 1

source_name: synthetic DNA oligos


title: fcp1-452_+N_37_rep1 (set 2)

source_name: fcp1-452 mutant strain, incubated in EMM2 at 37ºC

tag: fcp1-452 mutant strain, background :L972 h- wild type, backcrossed with h+ wild type


title: WT_+N_37 (set 1)


title: WT_+N_37_rep2 (set 2)


title: fcp1-452_+N_37_rep2 (set 2)


title: WT_-N_26 (set 1)

source_name: L972, incubated in EMM2-N at 26ºC


title: fcp1-452_+N_26_rep2 (set 2)

source_name: fcp1-452 mutant strain, logarithmically growing in EMM2 at 26ºC


title: fcp1-452_+N_26_rep1 (set 2)


title: fcp1-452_+N_26 (set 1)


title: WT_-N_37 (set 1)

source_name: L972, incubated in EMM2-N at 37ºC


title: WT_+N_26 (set 1)


title: O.cincta Cd tolerant vs Lab culture_cadmium_3

status: Public on Apr 01 2009

city: Amsterdam

first: Dick

last: Roelofs

state: Noord-Holland

address: De Boelelaan 1085

department: Animal Ecology

country: Netherlands

institute: VU University

description:
Full hybridisation protocol:
Hybridization of labeled cDNA to oligonucleotide-DNA on CodeLink slides using HybArray12
Materials, solutions and buffers
• Salmon sperm DNA (10 μg/μl, Invitrogen, 1563-011)
• Poly A (pd(A)40-60,10 μg/μl, Amersham, 27-798801)
• tRNA (9.2 μg/μl, Sigma, R-5636)
• Cot1 DNA (1.0 μg/μl, Invitrogen,15279-011)
• ethanol absolute
• 10% SDS (Sigma, L-4522)
• 0.2X SSC
• 0.1X SSC
• 0.01X SSC
• Master Mix:
− 1 g dextran sulphate (USB, US 70796)
− 5.3 ml formamide 100% (Invitrogen,15515-026)
− 0.7 ml MilliQ
− 1.0 ml 20X SSC (Sigma, S-6639)
− pH 7.0
• Washing buffer (formamide/SSC):
− 50% formamide (Fluka, 47670)
− 2X SSC
− pH 7.0
• PN-buffer:
− 0.1 M Na2HPO4 /0.1 M NaH2PO4
− 0.1% Igepal CA630 (Sigma, I-7771)
− pH 8.0
Blocking
Before applying (pre)hybridization mix block slides according to the Blocking Codelink Activated Slides protocol.
Preparation of (pre)hybridization mix for HybArray12
Download here the Excel template to calculate for multiple slides.
Pre-Hybridization mix:
• for preparation of 126.7 μl prehybridization mix (one slide), mix together:
− 3.0 μl (30 μg ) Salmon sperm DNA
− 1.2 μl (12 μg ) poly A
− 6.5 μl (60 μg) tRNA
− 24 μl (24 μg) Cot1 DNA
• precipitate using 0.1 vol. 3 M NaAc pH 5.2 and 2.5 vol. ethanol absolute, incubate on ice for 5 min, spin down 10 min at 12000 g and 4°C
• wash pellet with 250 μl 70% ethanol, spin for 5 min at 12000 g and 4°C
• air dry pellet until no ethanol visible
• dissolve pellet in 40.7 μl MilliQ by pipetting
• add 2.5 μl of 10% SDS
• add 83.5 μl of Master-Mix
• gently dissolve the mixture
Hybridization mix:
• for preparation of 126.7 μl hybridization mix (one slide), mix together:
− 120 μl of labelled cDNA (combine 60 μl Cy3 and 60 μl Cy5 probe)
− 1.2 μl (12 μg ) poly A
− 6.5 μl (60 μg) tRNA
− 24 μl (24 μg) Cot1 DNA
• precipitate using 0.1 vol. 3 M NaAc pH 5.2 (15.2 μl) and 2.5 vol. ethanol absolute (380 μl), incubate on ice for 5 min, spin down 10 min at 12000 g and 4°C
• wash pellet with 250 μl 70% ethanol, spin for 5 min at 12000 g and 4°C
• air dry pellet until no ethanol visible
• dissolve pellet in 40.7 μl MilliQ by pipetting
• add 2.5 μl of 10% SDS
• add 83.5 μl of Master-Mix
• gently dissolve the mixture
• incubate for 10 min at 70°C
• 1 min on ice/water
• incubate 1 hour at 37°C
Hybridization on HybArray 12
For working with the HybArray you have to book your time (in-house only).
DO NOT use the HybArray without instructions from qualified personnel!!!!
• choose your hybridization unit (1 to 6, two slides per unit, as specified on the website)
• assemble the hybridization unit:
− insert rubber O-rings in the cover
− black bottom plate: put the slide in the proper direction! Make sure you can read the Code-Link text. Also this text must be loaded on the upper side of the unit.
• introduce the unit in the HybArray12
• insert plugs
• insert tubes in corresponding wash bottles
• choose:
− start a run
− from floppy
− prehyb.hyb
− load
− select slides
• when the Hyb-station is ready (visible on screen) apply prehyb mix:
− press Probe to add the prehyb. for the selected slide
− check if a mark on the screen appears
− take the plug out and inject the prehyb. by pipetting it slowly into the port using a 200 ul pipet
− press the Finished control (check mark) and insert the plug
− repeat this for the next slide
− press the Finished control for the selected slide
• after 45 minutes take out the slides and put them in 0.01 x SSC
• centrifuge slides for 3 minutes, 1000 rpm, 20°C (in 50 ml tube)
• reassemble hybridization unit (make sure cover is completely dry!)
• introduce the unit in the station
• insert plugs
• choose:
− start a run
− from floppy
− oligo.hyb
− load
− select slides
• apply the hybridization mix (probe) at the same way as the prehyb. solution
• take slides out next morning (or 14 hours later) and put them in 0.01 x SSC
• centrifuge slides for 3 minutes, 1000 rpm, 20°C (in 50 ml tube)
• slides are ready for scanning
• reassemble the hybridization unit with dummy slides
• put all the tubes in MilliQ
• choose:
− maintenance
− Machine Cleaning Cycle
• When machine is finished, take out the hybridization unit, rinse everything with MilliQ, especially the sample port and leave it at the sink to dry (take out rubber rings)
Programs for the HybArray 12
Prehyb.hyb:
Step 1. Introducing prehybribization solution
Temperature 37°C
2. Set Slide Temperature
Temp:37 Time:45m:00s Agitate:Yes
3. Drain Slides
Waste Bottle1. Time 1m:00s
Oligo.hyb:
Step 1. Introducing prehybribization solution
Temperature 37°C
2. Set Slide Temperature
Temp:37 Time:14h:00s Agitate:Yes
3. Wash slides 50% formamide/2X SSC at 35°C; 6 cycles, flow 10 sec, hold 20 sec
4. Wash slides PN-buffer at 25°C ; 2 cycles, flow 10 sec, hold 20 sec 5. Wash slides 0.2X SSC at 25°C 2 cycles, flow 10 sec, hold 20 sec
6. Wash slides 0.1X SSC; at 25°C 2 cycles, flow 10 sec, hold 20 sec
7. Wash slides 0.01X SSC; at 25°C 1 cycles, flow 10 sec, hold 20 sec

hyb_protocol: Hybridization of labeled cDNAs was performed according to Bergman et al. (Bergman AM, Eijk PP, van Haperen V, et al. (2005) In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant. Cancer Research 65, 9510-9516) with a modified hybridization buffer (0.14 g/ml dextrane sulfate, 75% formamide, 3XSSC, 60 μg t-RNA and 24 μg human cot-1 DNA, Amersham) at 37 ˚C in a HybArray 12 (Perkin Elmer) for 14 hours. Wash steps were also performed in the HybArray 12 and included one step with 50% formamide/2XSSC, one step with phosphate buffer followed by three steps with SSC (0.2X, 0.1X, and 0.01X).

scan_protocol: Labeled cDNAs bound on microarray slides were quantified with a DNA Microarray Scanner (Agilent, US). Scan settings Scan region Scan Area (61 x 21.6 mm) Scan resolution (μm) 5 5μm scanning mode Single Pass eXtended Dynamic range selected Dye channel Red & Green Green PMT XDR Hi 100% XDR Lo 10% Red PMT XDR Hi 100% XDR Lo 10% Split and Rotate (default setting) Split and Rotate OFF

data_processing: Images were analyzed using Bluefuse microarray analysis software (version 3.2, BlueGnome, UK, http://www.cambridgebluegnome.com/bluefuse.htm) in order to calculate background corrected log2 gene expression ratios of Cy5/Cy3 signals.

organism: Orchesella cincta

source_name: Plombieres culture, cadmium treatment, 3 day exposure

label_protocol: 20 μg of total RNA was labeled by the incorporation of 5-(3-aminoallyl)-dUTP (Ambion) during cDNA synthesis with superscript II reverse transcriptase (Invitrogen) and an oligo(d)T15 probe. Subsequently, amine-labeled cDNA was treated with Fluorolink Cy-3 monofunctional dye (Amersham). After removal of unincorporated fluorescent dyes, with a PCR clean up column (Qiagen), labeled cDNA was eluted in nuclease free water and stored at -80˚ C until hybridization.

growth_protocol: To diminish putative environmental effects from the field the animals were reared in a climate room (20°C, 75% humidity, LD 12 h:12 h) in PVC jars on a moist plaster of Paris layer feeding on algae present on twigs for at least three generations before experimental treatment.

extract_protocol: Total RNA was extracted from all animals per jar, using the Tissue extraction protocol of the SV Total RNA isolation System (Promega). PDF available at: http://www.promega.com/tbs/tm048/tm048.html

treatment_protocol: 3 day exposure after moulting in a jar containing algae food with 1 micromole cadmium/gram dry algae

tag: Pool of 12 animals (approximately 30 days old), cadmium tolerant culture derived from Plombieres population.

source_name: Lab culture, cadmium treatment, 3 day exposure

label_protocol: 20 μg of total RNA was labeled by the incorporation of 5-(3-aminoallyl)-dUTP (Ambion) during cDNA synthesis with superscript II reverse transcriptase (Invitrogen) and an oligo(d)T15 probe. Subsequently, amine-labeled cDNA was treated with Fluorolink Cy-5 monofunctional dye (Amersham). After removal of unincorporated fluorescent dyes, with a PCR clean up column (Qiagen), labeled cDNA was eluted in nuclease free water and stored at -80˚ C until hybridization.

tag: Pool of 12 animals (approximately 30 days old), Lab culture derived from Roggebotzand reference population.


title: O.cincta Cd tolerant vs Lab culture_cadmium_4


title: fcp1-452_-N_26 (set 1)

source_name: fcp1-452 mutant strain,  incubated in EMM2-N at 26ºC


title: O.cincta Cd tolerant_cadmium_4

source_name: Plombieres culture, reference, 3 day exposure

treatment_protocol: 3 day exposure after moulting in a jar containing clean algae food


title: fcp1-452_-N_37 (set 1)

source_name: fcp1-452 mutant strain,  incubated in EMM2-N at 37ºC


title: O.cincta Cd tolerant vs Lab culture_clean_1

source_name: Lab culture, reference, 3 day exposure


title: O.cincta Cd tolerant vs Lab culture_clean_2


title: O.cincta Cd tolerant vs Lab culture_clean_4


title: O.cincta Cd tolerant vs Lab culture_clean_3


title: O.cincta Cd tolerant_cadmium_1


title: O.cincta Cd tolerant vs Lab culture_cadmium_2


title: O.cincta Cd tolerant_cadmium_2


title: O. cincta lab culture_cadmium_2


title: O.cincta Cd tolerant_cadmium_3


title: O. cincta lab culture_cadmium_1

description:
Full hybridisation protocol:
Hybridization of labeled cDNA to oligonucleotide-DNA on CodeLink slides using HybArray12
Materials, solutions and buffers
• Salmon sperm DNA (10 μg/μl, Invitrogen, 1563-011)
• Poly A (pd(A)40-60,10 μg/μl, Amersham, 27-798801)
• tRNA (9.2 μg/μl, Sigma, R-5636)
• Cot1 DNA (1.0 μg/μl, Invitrogen,15279-011)
• ethanol absolute
• 10% SDS (Sigma, L-4522)
• 0.2X SSC
• 0.1X SSC
• 0.01X SSC
• Master Mix:
− 1 g dextran sulphate (USB, US 70796)
− 5.3 ml formamide 100% (Invitrogen,15515-026)
− 0.7 ml MilliQ
− 1.0 ml 20X SSC (Sigma, S-6639)
− pH 7.0
• Washing buffer (formamide/SSC):
− 50% formamide (Fluka, 47670)
− 2X SSC
− pH 7.0
• PN-buffer:
− 0.1 M Na2HPO4 /0.1 M NaH2PO4
− 0.1% Igepal CA630 (Sigma, I-7771)
− pH 8.0
©VUmc Micro Array Facility, April 2004
Hybridization of labeled cDNA to oligo-DNA targets on CodeLink slides using HybArray 12 5 pages
Blocking
Before applying (pre)hybridization mix block slides according to the Blocking Codelink Activated Slides protocol.
Preparation of (pre)hybridization mix for HybArray12
Download here the Excel template to calculate for multiple slides.
Pre-Hybridization mix:
• for preparation of 126.7 μl prehybridization mix (one slide), mix together:
− 3.0 μl (30 μg ) Salmon sperm DNA
− 1.2 μl (12 μg ) poly A
− 6.5 μl (60 μg) tRNA
− 24 μl (24 μg) Cot1 DNA
• precipitate using 0.1 vol. 3 M NaAc pH 5.2 and 2.5 vol. ethanol absolute, incubate on ice for 5 min, spin down 10 min at 12000 g and 4°C
• wash pellet with 250 μl 70% ethanol, spin for 5 min at 12000 g and 4°C
• air dry pellet until no ethanol visible
• dissolve pellet in 40.7 μl MilliQ by pipetting
• add 2.5 μl of 10% SDS
• add 83.5 μl of Master-Mix
• gently dissolve the mixture
Hybridization mix:
• for preparation of 126.7 μl hybridization mix (one slide), mix together:
− 120 μl of labelled cDNA (combine 60 μl Cy3 and 60 μl Cy5 probe)
− 1.2 μl (12 μg ) poly A
− 6.5 μl (60 μg) tRNA
− 24 μl (24 μg) Cot1 DNA
©VUmc Micro Array Facility, April 2004
Hybridization of labeled cDNA to oligo-DNA targets on CodeLink slides using HybArray 12 5 pages
• precipitate using 0.1 vol. 3 M NaAc pH 5.2 (15.2 μl) and 2.5 vol. ethanol absolute (380 μl), incubate on ice for 5 min, spin down 10 min at 12000 g and 4°C
• wash pellet with 250 μl 70% ethanol, spin for 5 min at 12000 g and 4°C
• air dry pellet until no ethanol visible
• dissolve pellet in 40.7 μl MilliQ by pipetting
• add 2.5 μl of 10% SDS
• add 83.5 μl of Master-Mix
• gently dissolve the mixture
• incubate for 10 min at 70°C
• 1 min on ice/water
• incubate 1 hour at 37°C
Hybridization on HybArray 12
For working with the HybArray you have to book your time (in-house only).
DO NOT use the HybArray without instructions from qualified personnel!!!!
• choose your hybridization unit (1 to 6, two slides per unit, as specified on the website)
• assemble the hybridization unit:
− insert rubber O-rings in the cover
− black bottom plate: put the slide in the proper direction! Make sure you can read the Code-Link text. Also this text must be loaded on the upper side of the unit.
• introduce the unit in the HybArray12
• insert plugs
• insert tubes in corresponding wash bottles
• choose:
− start a run
− from floppy
− prehyb.hyb
− load
− select slides
©VUmc Micro Array Facility, April 2004
Hybridization of labeled cDNA to oligo-DNA targets on CodeLink slides using HybArray 12 5 pages
• when the Hyb-station is ready (visible on screen) apply prehyb mix:
− press Probe to add the prehyb. for the selected slide
− check if a mark on the screen appears
− take the plug out and inject the prehyb. by pipetting it slowly into the port using a 200 ul pipet
− press the Finished control (check mark) and insert the plug
− repeat this for the next slide
− press the Finished control for the selected slide
• after 45 minutes take out the slides and put them in 0.01 x SSC
• centrifuge slides for 3 minutes, 1000 rpm, 20°C (in 50 ml tube)
• reassemble hybridization unit (make sure cover is completely dry!)
• introduce the unit in the station
• insert plugs
• choose:
− start a run
− from floppy
− oligo.hyb
− load
− select slides
• apply the hybridization mix (probe) at the same way as the prehyb. solution
• take slides out next morning (or 14 hours later) and put them in 0.01 x SSC
• centrifuge slides for 3 minutes, 1000 rpm, 20°C (in 50 ml tube)
• slides are ready for scanning
• reassemble the hybridization unit with dummy slides
• put all the tubes in MilliQ
• choose:
− maintenance
− Machine Cleaning Cycle
• When machine is finished, take out the hybridization unit, rinse everything with MilliQ, especially the sample port and leave it at the sink to dry (take out rubber rings)
©VUmc Micro Array Facility, April 2004
Hybridization of labeled cDNA to oligo-DNA targets on CodeLink slides using HybArray 12 5 pages
©VUmc Micro Array Facility, April 2004
Programs for the HybArray 12
Prehyb.hyb:
Step 1. Introducing prehybribization solution
Temperature 37°C
2. Set Slide Temperature
Temp:37 Time:45m:00s Agitate:Yes
3. Drain Slides
Waste Bottle1. Time 1m:00s
Oligo.hyb:
Step 1. Introducing prehybribization solution
Temperature 37°C
2. Set Slide Temperature
Temp:37 Time:14h:00s Agitate:Yes
3. Wash slides 50% formamide/2X SSC at 35°C; 6 cycles, flow 10 sec, hold 20 sec
4. Wash slides PN-buffer at 25°C ; 2 cycles, flow 10 sec, hold 20 sec 5. Wash slides 0.2X SSC at 25°C 2 cycles, flow 10 sec, hold 20 sec
6. Wash slides 0.1X SSC; at 25°C 2 cycles, flow 10 sec, hold 20 sec
7. Wash slides 0.01X SSC; at 25°C 1 cycles, flow 10 sec, hold 20 sec


title: O. cincta lab culture_cadmium_3


title: O.cincta Cd tolerant vs Lab culture_cadmium_1


title: O. cincta lab culture_cadmium_4


title: fcp1-452_+N_37 (set 1)


title: NPIP biological replicate 1

city: Maastricht

first: Dennie

last: Hebels

address: Universiteitssingel 50

department: Health Risk Analysis and Toxicology

institute: Maastricht University

description: Biological replicate 1 of 4

hyb_protocol: Hybridization according to Agilent instructions.

scan_protocol: Slides were scanned on a GenePix® 4000B Microarray Scanner (Molecular Devices, Sunnyvale, USA). Cy3 and Cy5 were excited at wavelengths of 532 and 635 nm, respectively. Laser power was set to 100%. The photo multiplier tube gain was set to a saturation tolerance of 0.02% to minimize background and saturated spots. Photomultiplier gain was set to 587 (Cy3) and 705 (Cy5) for replicates 1 and 2, and 628 (Cy3) and 560 (Cy5) for replicates 3 and 4, based on automatic establishment of ideal scan settings. The images obtained (resolution 5 micron, 16 bit tiff image) were processed with Imagene 8.0.1 software (Biodiscovery, El Segundo, USA) to measure mean signal intensities for spots and local backgrounds followed by a quality control in Microsoft Excel.

data_processing: Data preparations were performed in GeneSight software (BioDiscovery) and included in chronological order: background subtraction, omission of bad spots (controls and irregularly shaped spots), LOWESS normalization, log (base=2) transformations, calculation of difference of test compound versus respective vehicle control.

source_name: Control for NPIP 24h

label_protocol: The Two-Color Microarray-Based Gene Expression Analysis kit from Agilent Technologies (Amstelveen, The Netherlands) was used to generate Cyanine (Cy) labelled cRNA according to the manufacturer’s protocol.

growth_protocol: Caco-2 cell cultures were transferred weekly by trypsinization and incubated at 37 ºC in a humidified incubator containing 5% CO2

extract_protocol: RNA was isolated from TRIzol® suspended cells according to the manufacturer’s protocol with minor modifications, followed by a clean up, using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) with DNase treatment.

treatment_protocol: Caco-2 cells were treated for 24 hours with one of the following six NOC: MNNG (1μM), MNU (1mM), NDEA (50mM), NDMA (100mM), NPIP (40mM) and NPYR (100mM)

tag: Caco-2 (Human colonic adenocarcinoma cells)

source_name: NPIP 40mM 24h


title: NPYR biological replicate 3

description: Biological replicate 3 of 4

source_name: Control for NPYR 24h

source_name: NPYR 100mM 24h


title: NPYR biological replicate 2

description: Biological replicate 2 of 4


title: NPIP biological replicate 4

description: Biological replicate 4 of 4


title: NPIP biological replicate 3

description: Biological replicate 3 of 4


title: NDMA biological replicate 1

description: Biological replicate 1 of 4

source_name: Control for NDMA 24h

source_name: NDMA 100mM 24h


title: NDEA biological replicate 3

description: Biological replicate 3 of 4

source_name: Control for NDEA 24h

source_name: NDEA 50mM 24h


title: NPIP biological replicate 2

description: Biological replicate 2 of 4


title: NDEA biological replicate 1

description: Biological replicate 1 of 4


title: NDEA biological replicate 4

description: Biological replicate 4 of 4


title: NDMA biological replicate 2

description: Biological replicate 2 of 4


title: NDMA biological replicate 3

description: Biological replicate 3 of 4


title: NDEA biological replicate 2

description: Biological replicate 2 of 4


title: NPYR biological replicate 4

description: Biological replicate 4 of 4


title: MNU biological replicate 2

description: Biological replicate 2 of 4

source_name: MNU 1mM 24h

source_name: Control for MNU 24h


title: MNU biological replicate 1

description: Biological replicate 1 of 4


title: NDMA biological replicate 4

description: Biological replicate 4 of 4


title: NPYR biological replicate 1

description: Biological replicate 1 of 4


title: MNNG biological replicate 4

description: Biological replicate 4 of 4

source_name: MNNG 1μM 24h

source_name: Control for MNNG 24h


title: Sall4_KO_MEFminus

city: Kumamoto

first: Ryuichi

last: Nishinakamura

address: 2-2-1 Honjo

department: Institute of Molecular Embryology and Genetics

institute: Kumamoto University

description: Sall4 null (delta/-) ES cells were electroporated with a targeting vector containing Oct3/4 genomic regions and an IRES-blasticidin-resistance gene (BSD) and cultured on gelatin-coated dish.

hyb_protocol: The protocol and conditions used for hybridization, blocking and washing: 10 micrograms of fragmented cRNA were hybridised (45 degrees Celsius 16 hours). Hybridization was controlled by use of the GeneChip Eukaryotic Hybridization Control Kit (Affymetrix). Washing and staining was performed in a Fluidics Station 450 (Affymetrix) using the protocol EukGE-WS2v4.

scan_protocol: Scanning was performed in an Affymetrix GeneChip scanner 3000 7G.

data_processing: Chip analysis was performed using the Affymetrix Microarray Suite v5.0. MAS5-calculated Signal intensity (target intensity=100)

source_name: Sall4-null (KO1) ES cells, minus feeder cells

label_protocol: In vitro transcription was performed on 1 microgram of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc.

growth_protocol:
Growth medium : ES cell Medium - GMEM (high glucose)
Media supplements: FCS (heat inactivated), non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, LIF, Blasticidin
Culture conditions : 37C, 5% CO2

extract_protocol: Total RNAs were extracted using Isogen (Invitrogen). 3 micrograms of total RNA was reverse transcribed using the SuperScript III system for cDNA synthesis (Invitrogen) according to the protocol recommended by Affymetrix. The oligonucleotide used for priming was 5'-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3' (Invitrogen). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf).

tag: Strain

value: E14.1 ES cell

value: delta/-

tag: Feeder cells

value: absent


title: MNNG biological replicate 2

description: Biological replicate 2 of 4


title: MNNG biological replicate 3

description: Biological replicate 3 of 4


title: Sall4_KO_MEFplus

description: Sall4 null (delta/-) ES cells were electroporated with a targeting vector containing Oct3/4 genomic regions and an IRES-blasticidin-resistance gene (BSD) and cultured on blasticidin-resistant fibroblasts.

source_name: Sall4-null (KO1) ES cells, plus feeder cells

value: present


title: MNU biological replicate 4

description: Biological replicate 4 of 4


title: MNU biological replicate 3

description: Biological replicate 3 of 4


title: Sall4_HET_MEFminus

description: Sall4 heterozygous (flox/-) ES cells were electroporated with a targeting vector containing Oct3/4 genomic regions and an IRES-blasticidin-resistance gene (BSD) and cultured on gelatin-coated dish.

source_name: Sall4 heterozygous (HET18) ES cells, minus feeder cells

value: flox/-


title: Sall4_HET_MEFplus

description: Sall4 heterozygous (flox/-) ES cells were electroporated with a targeting vector containing Oct3/4 genomic regions and an IRES-blasticidin-resistance gene (BSD) and cultured on blasticidin-resistant fibroblasts.

source_name: Sall4 heterozygous (HET18) ES cells, plus feeder cells


title: MNNG biological replicate 1

description: Biological replicate 1 of 4


title: NCFM vs. upp mutant_OD 0.8_Rep B

status: Public on Mar 24 2009

city: Raleigh

first: Todd

middle: Robert

last: Klaenhammer

state: NC

department: Department of Food, Bioprocessing, and Nutrition Sciences

institute: North Carolina State University

description: Signal intensities (stored as TIFF image files) were quantified, the background was subtracted, and data were normalized by using the QuantArray 3.0 software package (Perkin-Elmer).

hyb_protocol: Prior to hybridization, oligoarray slides were prehybridized in a buffer containing 0.1% (wt/vol) SDS (Invitrogen), 0.1% (wt/vol) bovine serum albumin (Invitrogen), and 5X SSC (1X SSC is 0.15 M NaCl plus 0.015 M sodium citrate; Invitrogen) at 42 C for 1 hr, then rinsed by submerging 5 times in sterile distilled water, twice in isopropanol, and spin-dry.  Pairwise Cy3- and Cy5-labeled probes were dried with a Vacufuge speed-vacuum system (Epperdorf, Westbury, NY) and resuspended in a total volume of 48-50 µl of hybridization buffer containing 5X SSC, 5X Denhardt’s solution (Invitrogen), 30% (vol/vol) formamide, 0.1% SDS, and 200 µg/ml of salmon sperm DNA (Invitrogen).  The resulting probe mixtures were incubated at 95°C for 2 min and hybridized to the oligoarrays for 18-20 hrs at 42 C.  Post-hybridized arrays were first washed in pre-warmed (42 C) washing buffer containing 1X SSC and 0.03% SDS, followed by a second wash in washing buffer containing 0.5X SSC and 0.03% SDS at room temperature.  Subsequently, the arrays were washed in 0.2X SSC followed by a final wash in 0.05X SSC.  Each washing step was performed for 5 min.

scan_protocol: Fluorescence intensities were acquired at 10-µm per pixel resolution by using a ScanArray 4000 Microarray Scanner (Packard Biochip BioScience; Biochip Technologies LLC) and stored as TIFF image files.

data_processing: Spots were analyzed by adaptive quantitation. The data were median normalized (Azcarate-Peril et al., 2005 Appl. Environ. Microbiol. 71:5794-804).  The Cy5/Cy3 ratio from 12 spots for each gene (6 replicated spots per slide per biological replicate) was averaged and represented the change (n-fold) in gene expression levels in the upp mutant (Cy5) relative to the NCFM (Cy3).  Confidence intervals and P values for the fold change were also calculated by using two-sample t-test.  P values of 0.05 or less were considered significant.

organism: Lactobacillus acidophilus NCFM

source_name: NCFM, mid-logarithmic phase (OD600nm 0.8)

label_protocol: Reverse transcription and fluorescent labeling of identical amounts (20 µg) of purified RNA was performed with the SuperScript™ Indirect cDNA Labeling System for DNA Microarrays (Invitrogen, Carlsband, CA) according to the manufacturer’s directions except 6 µg of random primers (Invitrogen) were used for each 30 µl in the first strand cDNA synthesis reaction.  Labeled cDNA samples were coupled with mono-reactive Cy3 or Cy5 dyes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).

extract_protocol: NCFM was grown in fresh MRS medium with a 2% (vol/vol) inoculum from overnight culture.  Aliquots of cells were collected at OD600 0.8 (mid-log) by quick centrifugation at room temperature.  Cell pellets were flash-frozen in ethanol-dry ice bath and stored at -80 C.  Samples were collected from two independent experimental replicates (representing two biological replicates, A and B).  Total RNA isolation, DNase-treatment and RNA purification were performed essentially as described previously (Goh et al., 2006 Appl. Environ. Microbiol. 72:7518-30).

tag: Total RNA was collected from cells grown in MRS medium until OD600nm reached 0.8

source_name: NCFM upp mutant (NCK1909), mid-logarithmic phase (OD600nm 0.8)

extract_protocol: NCFM upp mutant strain was grown in fresh MRS medium with a 2% (vol/vol) inoculum from overnight culture.  Aliquots of cells were collected at OD600 0.8 (mid-log phase) by quick centrifugation at room temperature.  Cell pellets were flash-frozen in ethanol-dry ice bath and stored at -80 C.  Samples were collected from two independent experimental replicates (representing two biological replicates, A and B).  Total RNA isolation, DNase-treatment and RNA purification were performed essentially as described previously (Goh et al., 2006 Appl. Environ. Microbiol. 72:7518-30).


title: NCFM vs. upp mutant_OD 0.3_Rep B

source_name: NCFM, early logarithmic phase (OD600nm 0.3)

extract_protocol: NCFM was grown in fresh MRS medium with a 2% (vol/vol) inoculum from overnight culture.  Aliquots of cells were collected at OD600 0.3 (early log-phase) by quick centrifugation at room temperature.  Cell pellets were flash-frozen in ethanol-dry ice bath and stored at -80 C.  Samples were collected from two independent experimental replicates (representing two biological replicates, A and B).  Total RNA isolation, DNase-treatment and RNA purification were performed essentially as described previously (Goh et al., 2006 Appl. Environ. Microbiol. 72:7518-30).

tag: Total RNA was collected from cells grown in MRS medium until OD600nm reached 0.3

source_name: NCFM upp mutant (NCK1909), early logarithmic phase (OD600nm 0.3)

extract_protocol: NCFM upp mutant strain was grown in fresh MRS medium with a 2% (vol/vol) inoculum from overnight culture.  Aliquots of cells were collected at OD600 0.3 (early log-phase) by quick centrifugation at room temperature.  Cell pellets were flash-frozen in ethanol-dry ice bath and stored at -80 C.  Samples were collected from two independent experimental replicates (representing two biological replicates, A and B).  Total RNA isolation, DNase-treatment and RNA purification were performed essentially as described previously (Goh et al., 2006 Appl. Environ. Microbiol. 72:7518-30).


title: NCFM vs. upp mutant_OD 0.3_Rep A


title: NCFM vs. upp mutant_OD 0.8_Rep A


title: CD38-CD49d- 14062_3

status: Public on Mar 03 2009

city: Aviano

first: daniela

last: marconi

address: Via franco Gallini 2

country: Italy

institute: CRO AVIANO

description: negative expression of CD38-CD49 in CLL cells

hyb_protocol: The Hybridization was performed acording to Ambion protocol

scan_protocol: slides were analysed by Agilent Microarray Scanner G2565AA

data_processing:
Background subtraction method: Marginal log-likelihood of foreground
values for normal + exponential model and its derivatives
Whithin Normalization method: loess
Between Normalization method: quantile

source_name: cd39-cd49 negative

label_protocol:
Total RNA was labelled according to Ambion protocol. 825 ng
Cy5-labeled aRNA from each CLL was hybridized (according to Agilent
protocol)

extract_protocol: Total RNA was extracted according to TRIZOL reagent protocol

tag: aRNA from purified CLL cells

source_name: reference

label_protocol:
Total RNA was labelled according to Ambion protocol. 825 ng of
Cy3-labeled reference aRNA (according to Agilent protocol)

tag: aRNA from pooled normal PB B cells of healthy donors


title: CD38+CD49d+ 10257_4

source_name: cd39-cd49 positive


title: CD38+CD49d+ 10163_3


title: Flt3 TKD+MLL-AF4 expressing 32Dc

status: Public on Mar 31 2009

city: Tokyo

first: Hiroki

last: Yamaguchi

address: 1-1-5 Sendagi, Bunkyo-Ku

department: Division of Hematology, Department of Internal Medicine

institute: Nippon Medical School

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Mouse Genome 430 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using GeneChip Scanner 3000 7G.

data_processing: Single Array Analysis were calculated by  Microarray Suite version 5.0 (MAS5.0) with Affymetrix default setting and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

source_name: Flt3 TKD+MLL-AF4 expressing 32Dc

label_protocol: cDNA were synthesized by GeneChip T7-Oligo(dT) Promoter Primer Kit（Affymetrix) and TaKaRa cDNA Synthesis Kit （TaKaRa Bio） from 3ug total RNA. Biotinylated cRNA were synthesized by IVT Labeling Kit(Affymetrix)

extract_protocol: Total RNA was extracted using Trizol Reagent (invitrogen) according to manufacturer's instructions and purified on RNeasy MinElute Spin Columns (Qiagen).

tag: cell line


title: 32DC


title: CD38+CD49d+ 14064_1


title: CD38+CD49d+ 10255_3


title: CD38+CD49d+ 10163_4


title: MLL-AF4 expressing 32Dc

source_name: MLL-AF4 expressing 32Dc


title: CD38+CD49d+ 10161_1


title: CD38-CD49d- 14063_3


title: CD38+CD49d+ 10256_4


title: CD38+CD49d+ 10162_2


title: CD38+CD49d+ 10139_4


title: CD38-CD49d- 14062_4


title: CD38-CD49d- 14064_3


title: CD38+CD49d+ 10162_1


title: CD38-CD49d- 14063_2


title: CD38-CD49d- 10258_4


title: CD38+CD49d+ 10162_4


title: CD38-CD49d- 14064_2


title: CD38-CD49d- 14063_4


title: CD38-CD49d- 10258_3


title: CD38-CD49d- 10176_2


title: CD38-CD49d- 10163_2


title: CD38-CD49d- 10176_3


title: CD38-CD49d- 10174_4


title: A427 cancer cell line sample CGH

type: genomic

city: New Haven

first: Pedro

middle: P

last: Medina-Vico

state: CT

address: 266 Whitney Ave, 938 KBT

institute: Yale University

description: The goal of this analysis is to see the copy number genes differential changed in lung cancer cell lines in relation with a control (normal females).

hyb_protocol: 3.- Hybridization: the probe (mix Cy3-labeled and Cy5-labeled cDNAs) was centrifuged at full speed for 5min at room temperature. Then it was applied (approx. 44µl, leave 2µl from the bottom of the tube behind in order to avoid transferring any sediment to the slide) to a clean cover-slip and place on the pre-hybridized microarray. The slide was placed in a hybridization chamber with the printed area and the cover-slip at the top, and was incubated overnight (15-20h) at 55ºC in an oven.

scan_protocol: Slides were scanned for Cy3 and Cy5 following the steps: 1.- Place each slide in a cassette as follows: orient the slide so that it is array-side down and to the left; take cassette flat side down, place on flat surface and push down on the two buttons; with the buttons pressed, insert the slide (keeping everything flat); release buttons when slide has been fully inserted. 2.- Place cassettes into carousel. The etched side should now face to the left, and you should be looking at the back of the slide. Note: the cassette must start with the first slot. The scanner recognizes an empty slot as a sign to stop scanning. Close the scanner top. 3.- Double-click on the Agilent Scanner icon. When it opens, the scanner will go through some initialization steps. Also, if the lasers are not fully warm, there will be a warning sign that will give an approximate time that the lasers will be ready. 4.- Enter your name under Operator and check to see that the controls are set for the following: output directory is D:\niehs; both Red and Green channels are checked and are set to 100%; the size is 10 microns. 5.- Click on Options: choose to scan default slide area under Scan Region Options; specify slide area to largest area under Default Scan Region; click on Instrument serial number under Automatic File Naming (it will then be highlighted). Choose and type in your identifier for that day. Leave the Slot Number as is. 6.- Fill out log in the Scanner Cassette Log with your name, date and the order you put your slides into the carousel. This is important to assure proper renaming after scanning is complete. Click on OK to close the Options window. 7.- Double check that all the settings are properly set and click on Scan. 8.- After scan is complete, you will need to change the names to format pxxsxx. To do this: with the right mouse button, click on the Start menu and choose Explorer. Navigate your way through the files by clicking on the D drive on the far left hand side of the screen. Double click on D drive then double click onniehs. This folder contains all of the scanned files. Locate your slides by looking for the identifier you used for the scan. Click once to highlight name and click once more to get a cursor in the file name. Type in the proper name using the log you filled out earlier. Once all of the names have been changed, highlight the newest slides and move them into the appropriate boxes on Witchfire. 9.- Once you are done, log out of the computer, take the cassettes out of the carousel and your slides out of the cassettes. Turn off scanner.

data_processing: Cy3 and Cy5 fluorescent signals were quantified using GenePix Pro 5.0 (Axon Instruments Inc.). Substandard spots were manually flagged and discarded for further analysis. The Cy5/Cy3 ratios were normalized to the median ratio value using the DNMAD tool, based on a print-tip loess method. The conditions for the normalization were: 1) use negative flags, that means that the flagged spots are not used for the normalization and are turned into missing values; 2) do not use background correction. The normalized and semi-logarithmic transformed data were preprocessing using GEPAS software. Conditions for data pre-processing were: 1) merge replicates (the Cy5/Cy3 ratios of replicated spots were averaged); 2) filter missing values in order to exclude for further analysis any gene with data missing in more than 10% of the samples.

source_name: DNA from A427 labeled with Cy5

label_protocol:
1.- DNA from each sample were primed with 3µl of random hexamers d(N)6 0.5mg/ml (Promega) at 70ºC for 10min. For each labeling reaction, a master mix was prepared: 5µl First Strand buffer 5x, 2.5µl DTT 0.1M, 0.5µl unlabeled dNTPs 50x (dATP/dCTP/dGTP:dTTP 25:5), 1.5µl Cy5-dUTP 10mM or Cy3-dUTP 10mM (for Universal Human Reference RNA), 0.63µl RNasin Inhibitor and 1.25µl SuperScript II (200U/µl). The labeling reaction was incubated at room temperature for 10min and then incubated at 42ºC for 2h. Tubes were protected from direct light.
2.- Each fluorescent labeled cDNA was purified with CyScribe GFX Purification kit (Amersham) following manufacturer's instructions.
3.- The Cy3-labeled and Cy5-labeled cDNAs for each hybridization were combined into a single tube. 10µg of Human Cot-1 DNA (Invitrogen) were added.
4.- To precipitate each hybridization probe, 0.1 volumes of sodium acetate 0.1M and 2.5 volumes of absolute ethanol were added.
5.- Each probe was resuspended in 4µl of filtered EDTA 10mM ph 8 and denatured at 95ºC for 10min. 40µl of SlideHyb buffer 1x (Ambion), prewarmed at 65ºC, and blocking agents (mix of polyA and yeast tRNA, 10µg and 4µg in 2µl of final volume) were added.

growth_protocol: DMEM Glutamax, 10% FBS, Antibiotics (Peneciline, Streptomicine, Fungizone).

extract_protocol: Total DNA was extracted following conventional DNA extraction methods.

tag: Lung cancer cell line

source_name: Genomic DNA extracted from 5 females

extract_protocol: Genomic DNA extracted from blood using conventional protocols for DNA extraction.

tag: Genomic DNA extracted from blood


title: CD38-CD49d- 10139_2

data_processing:
Background subtraction method: Marginal log-likelihood of foreground values for normal + exponential model and its derivatives
Whithin Normalization method: loess
Between Normalization method: quantile

label_protocol: Total RNA was labelled according to Ambion protocol. 825 ng Cy5-labeled aRNA from each CLL was hybridized (according to Agilent protocol)


title: H441 cancer cell line sample CGH

source_name: DNA from H441 labeled with Cy5


title: H23 cancer cell line sample CGH

source_name: DNA from H23 labeled with Cy5


title: A549 cancer cell line sample CGH

source_name: DNA from A549 labeled with Cy5


title: H1299 cancer cell line sample CGH

source_name: DNA from H1299 labeled with Cy5


title: H522 Lung cancer cell line CGH

source_name: DNA from H522 cell line labeled with Cy5


title: Calu3 cancer cell line sample

description: The goal of this analysis is to see the genes differential expressed in the lung cancer cell line in relation with a control (universal RNA Stratagene).

source_name: RNA from Calu3 labeled with Cy5

label_protocol:
1.- RNA from each sample were primed with 3µl of random hexamers d(N)6 0.5mg/ml (Promega) at 70ºC for 10min. For each labeling reaction, a master mix was prepared: 5µl First Strand buffer 5x, 2.5µl DTT 0.1M, 0.5µl unlabeled dNTPs 50x (dATP/dCTP/dGTP:dTTP 25:5), 1.5µl Cy5-dUTP 10mM or Cy3-dUTP 10mM (for Universal Human Reference RNA), 0.63µl RNasin Inhibitor and 1.25µl SuperScript II (200U/µl). The labeling reaction was incubated at room temperature for 10min and then incubated at 42ºC for 2h. Tubes were protected from direct light.
2.- Each fluorescent labeled cDNA was purified with CyScribe GFX Purification kit (Amersham) following manufacturer's instructions.
3.- The Cy3-labeled and Cy5-labeled cDNAs for each hybridization were combined into a single tube. 10µg of Human Cot-1 DNA (Invitrogen) were added.
4.- To precipitate each hybridization probe, 0.1 volumes of sodium acetate 0.1M and 2.5 volumes of absolute ethanol were added.
5.- Each probe was resuspended in 4µl of filtered EDTA 10mM ph 8 and denatured at 95ºC for 10min. 40µl of SlideHyb buffer 1x (Ambion), prewarmed at 65ºC, and blocking agents (mix of polyA and yeast tRNA, 10µg and 4µg in 2µl of final volume) were added.

extract_protocol: Total RNA from lung tumors and normal lung tissues was isolated with  RNeasy mini kit (QIAGEN) and digestion with RNase-free DNase I (QIAGEN)  following the manufacturer's instructions. Universal Human Reference RNA  (Stratagene), the reference used in all the hybridizations, is provided in a  solution of 70% ethanol and 0.1M sodium acetate; it was prepared by centrifugation and washing with 70% ethanol.

source_name: RNA from Stratagene's Universal Human Reference RNA labeled with Cy3

extract_protocol: Commercial sample already extracted.

tag: Stratagene’s Universal Human Reference RNA is composed of total RNA from 10 human cell lines. The reference RNA is designed to be used as a reference for microarray gene-profiling experiments. Equal quantities of DNase-treated total RNA from each cell line (adenocarcinoma, mammary  gland; melanoma; hepatoblastoma, liver; liposarcoma; adenocarcinoma,  cervix; histiocytic lymphoma; macrophage; embryonal carcinoma, testis;  lymphoblastic leukemia, T lymphoblast; glioblastoma, brain; plasmacytoma;  myeloma; B lymphocyte) were pooled to make the Universal Human  Reference RNA.


title: H23 cancer cell line sample

source_name: RNA from H23 labeled with Cy5


title: A549 cancer cell line sample

source_name: RNA from A549 labeled with Cy5


title: CD38-CD49d- 10139_3