title: Young Control Day 3 #15 B

description:
II.	Experiment Description
1)	Experimental Design
·	Authors: J. Scott Pattison, Thomas Ellis Childs, Lillian Folk, Richard Madsen, and Frank W. Booth.
·	Laboratory: Dr. Frank Booth, University of Missouri-Columbia, 1600 E. Rollins St, Columbia, Mo, 65211
·	Contact: boothf@missouri.edu
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 1:
·	Experimental Factors: Age
·	How many hybridizations in the experiment: 45 unique samples were hybridized once per array x 3 arrays each = 135 separate hybridizations in total.
·	Experiment Description: To elucidate the differences in gene expression between young control soleus muscles and old control soleus muscles.
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Young Day 3 Control muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

Ø	Biomaterial manipulations
·	Growth conditions: normal
controlled environment
20-22 ºC average temperature
housed in guinea pig cages 2-3/cage
rubber mats covered the bottoms of cages
12/12 hour light/dark cycle
regular rat chow was provided ad libitum
water was available ad libitum
½” x 3” poplar dowel rods doused in apple juice were given for environmental enhancement
·	Treatments: N/A
·	Tissue handling: both soleus muscles from a single rat were excised, weighed, and immediately frozen in liquid N2, the muscles were then stored at –80ºC until RNA isolation.

Ø	Extract preparation
·	Extraction method: muscle tissue was powdered over liquid N2 then put into TRIzol solution and homogenized with a Polytron homogenizer for 3 bouts of 15 seconds on setting 7.  Total RNA was extracted as per manufacturer instructions.  Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturers recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).  Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-oligo(dT24) primer.  Following, second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 100-fold amplification of RNA.  Finally, the biotinylated populations of cRNA were fragmented according to the linked protocol.  A complete description of procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Minor modifications: All incubations were done in a thermocycler instead of a water bath, cDNA was quantified follwing cDNA synthesis, prior to in vitro transcription, using a PicoGreen kit (Molecular Probes). One microgram of cDNA was then in vitro transcribed.
·	Target Preparation: Target cDNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Quality control: The quality and amount of starting RNA, cDNA, cRNA, and fragmented cRNA were confirmed using agarose gel analyses.
·	Spike controls:  Briefly, spike controls were added to 10 µg fragmented cRNA before prior to hybridization. A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Hybridization Procedures
·	Hybridization:  Arrays were pre-hybridized, loaded, and hybridized for 16 hours prior to washing and staining.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx
·	Washing and Staining: Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Measurement data and specifications of data processing
·	Scanning:  The scanning protocols are available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Raw Image Analysis/Quality Control: After scanning, array images were assessed to confirm scanner alignment.  The corners were examined by eye for proper grid alignment.  Images were also scanned for the absence of bubbles, scratches, and speckling.  If any images with small scratches or speckles that skewed >25% of a given feature, that feature was masked and not quantitated in future analyses.  3'/5' ratios for GAPDH and beta-actin were confirmed to be ~1.0.  From the QC report, spike controls were found to be present on all chips, with BioB, BioC, BioD and CreX being found present in increasing intensity.
·	Scaling/Normalization: All data were scaled to a target intensity of 1500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background, and mean intensities.  No further normalization was applied.
·	Data Analysis:
·	Experiment 1:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a Student’s t-test.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 (raw p=4.478x10-6) were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)
·	Experiment 3:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 3-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)


title: Col_0_1

status: Public on Aug 06 2003

type: RNA

city: Tübingen

first: Markus

last: Schmid

address: Speemannstrasse 37-39

department: Molecular Biology

country: Germany

institute: Max Planck Institute for Developmental Biology

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 1
Keywords = flowering

molecule: total RNA

organism: Arabidopsis thaliana

source_name: shoot apex


title: CD8+ CM T cells (SubA)

status: Public on Jul 25 2003

city: Denver

first: Brad

last: Swanson

state: CO

country: USA

institute: National Jewish Medical and Research Center/HHMI

description:
•In vitro generated CD8+ central memory(CD8IL-15) t cells were generated from female P14 T cell receptor transgenic mice on the C57Bl/6 genetic background.

•Whole spleen and lymph node cells from female P14 T cell receptor transgenic mice were homogenized and stimulated with cognate peptide (KAVYNFATM) for 2 days.  The cells were then grown in IL-15 (central memory/CD8IL-15) for 7 additional days.  Central memory (CD8IL-15) CD8+ T cells were sorted by high speed cell sorting for the predominant CD62Lhigh population of cells.
Keywords = CD8, T cells, central memory

organism: Mus musculus

source_name: in vitro generated CD8+ central memory T cells


title: Col_0_2

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 2
Keywords = flowering


title: SG4 18BPFCy3 vs PFCy5 580650GEO

city: New Haven

first: Tong-Ruei

last: Li

state: CT

address: 333 Cedar St. Rm#NSB386

department: Department of Genetics

institute: Yale University School of Medicine

description:
The MIAME Checklist
Experiment Design:
·	Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between two time points (approximately 18 hours before pupariation staged according to Andres, A.J. & Thummel, C.S. Methods for quantitative analysis of transcription in larvae and prepupae. in Methods in Cell Biology, Volume 44.  Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994)) vs. at pupariation.  Abbreviations are: MG, midgut; SG, salivary gland; ED, epidermis with attached muscle; CNS, central nervous system; WD, wing disc.  RNA samples from tissues dissected from Drosophila melanogaster (Canton S strain) were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.  Samples from these two developmental stages were hybridized against each other on each microarray.
·	Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested differences in gene expression at different times in development in individual tissues.
·	The number of hybridizations performed in the experiment.
Three replicate hybridizations for each tissue using independent samples, except for wing disc samples, for which one sample pair were hybridized on 2 arrays.
·	The type of reference used for the hybridizations, if any.
NA
·	Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above
·	Quality control steps taken: for example, replicates or dye swaps.
Dye swaps were used only for CNS samples.  Three replicates for each tissue/organ.
·	URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
·	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.  See above.  Tissues/Organs were dissected from at least 12 individuals for each sample.
·	Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.  Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
·	Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using the SDS lysis protocol in Andres, A.J. & Thummel, C.S. “Methods for quantitative analysis of transcription in larvae and prepupae”. in  Methods in Cell Biology, Volume 44.  Drosophila melanogaster: Practical Uses in Cell and Molecular Biology., Vol. 44 (eds. Goldstein, L.S.B. & Fyrberg, E.A.) 565-573 (Academic Press, San Diego, 1994).  2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
·	Labeling protocol(s).
Priming step with random hexamers.  Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides.  Cleanup using Qiagen PCR cleanup kit.  Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker.  Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees.  Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry.  Arrays were stored in the dark until scanned.  See K.P. White and K.C. Burtis “Drosophila microarrays: from arrayer construction to hybridization” in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
·	External controls (spikes).
Hybridization procedures and parameters:
·	The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
·	The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
·	The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o	Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o	Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software.  The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o	The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o	Data selection and transformation procedures.
Data for all spots are filtered.  For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software.  (2). For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background.  (3). The net signal noise ratio should be at least 1 for at least one channel.  For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) to the background median.  The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot.  For the channel 532 nm wavelength, F532_median and B532_median are used.  Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper.  Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached.  Data before and after data selection is in one file.
Array Design:
·	General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome.  Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. “Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.” Nucleic Acids Research 29, e38 (2001).
·	For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files.  The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
·

organism: Drosophila melanogaster

source_name: 635nm (Cy5) channel, salivary gland at pupariation

source_name: 532nm (Cy3) channel, salivary gland at approximately 18 hours before pupariation


title: CD8+ E/EM T cells (SubA)

description:
•In vitro generated CD8+ effector/effector memory (CD8IL-2) T cells were generated from female P14 T cell receptor transgenic mice on the C57Bl/6 genetic background.

•Whole spleen and lymph node cells from female P14 T cell receptor transgenic mice were homogenized and stimulated with cognate peptide (KAVYNFATM) for 2 days.  The cells were then grown in IL-2 (effector/effector memory/CD8IL-2) for 7 additional days.  Effector/effector memory (CD8IL-2) CD8+ T cells were then sorted by high speed cell sorting for the predominant CD62Llow population of cells.

source_name: in vitro generated CD8+ effector/effector memory T cells


title: CNS2 PFCy3 vs 18BPFCy5 630660dyeswapGEO

source_name: 635nm (Cy5) channel, central nervous system at approximately 18 hours before pupariation

source_name: 532nm (Cy3) channel, central nervous system at pupariation


title: MG3 18BPFCy3 vs PFCy5 590660GEO

source_name: 635nm (Cy5) channel, midgut at pupariation

source_name: 532nm (Cy3) channel, midgut at approximately 18 hours before pupariation


title: WD1 18BPFCy3 vs PFCy5 800680GEO

source_name: 635nm (Cy5) channel, wing disc at pupariation, one of two hybridizations using sample No. 1

source_name: 532nm (Cy3) channel, wing disc at approximately 18 hours before pupariation, one of two hybridizations using sample No. 1


title: CNS3 18BPFCy3 vs PFCy5 630700GEO

source_name: 635nm (Cy5) channel, central nervous system at pupariation

source_name: 532nm (Cy3) channel, central nervous system at approximately 18 hours before pupariation


title: ED2 18BPFCy3 vs PFCy5 560650GEO

source_name: 635nm (Cy5) channel, epidermis with attached muscle at pupariation

source_name: 532nm (Cy3) channel, epidermis with attached muscle at approximately 18 hours before pupariation


title: E87b

status: Public on Jul 16 2003

city: Cambridge

first: Colin

last: Meiklejohn

state: MA

address: 16 Divinity Ave

department: Organismic and Evolutionary Biology

institute: Harvard University

description: Strains of Drosophila melanogaster were raised on standard medium at 25 C with no light/dark cycle.  Adult males were collected up to 24 hours after eclosing and separated from females where they were allowed to age an additional 3-4 days.  Total RNA was extracted by homogenizing 8-12 flies in TRIzol reagent (Invitrogen) followed by a chloroform extraction and isopropanol precipitation.  The resulting pellet was washed with 75% ethanol, air-dried, and resuspended in TE.  Individual extractions were pooled into one common RNA sample for each strain.  Poly-A RNA was purified from total RNA using the Oligotex Direct mRNA kit (Qiagen). mRNA samples were confirmed to be of high quality with a 2100 Bioanalyzer (Agilent).  The production of cDNA and hybridizations were done following a published protocol (Eisen and Brown, Methods in Enzymology 303:179-205).  2 µg of poly-A RNA was used as a template for SuperScript II reverse transcriptase (Invitrogen) in the presence of amino-allyl dUTP (Sigma) with random hexamer and oligo dT primers.  Reverse transcription was done at 42 C for 3 hours, followed by degradation of RNA by the addition of NaOH and EDTA and incubation at 65 C for 15 minutes.  The cDNA was concentrated and purified with Microcon-30 filters (Millipore).   Cyanine-3 or cyanine-5 fluorochromes (Amersham) were incorporated with the addition of 1 M NaHCO3 and a 75 minute incubation at room temperature with the cDNA probe.  The labeled probes were then purified using the QIAquick PCR Purification Kit (Qiagen), concentrated with Microcon-30 filters, and combined into one sample.  25 µL of probe was combined with 22 µg poly(A), 4.4 µL 20X SSC, 0.71 µL 1 M HEPES (pH 7.0), and 0.66 µL 10% SDS.  The probe was boiled at 100 C for 2 minutes and injected under a LifterSlip (Erie Scientific Company) placed over the array.  Slides were placed in a Telechem hybridization chamber with 3X SSC in the chamber wells to prevent drying, sealed, and placed in a 65 C water bath for 12 hours.  After hybridization, slides were washed with two wash solutions (0.6X SSC, 0.025% SDS [solution 1] and 0.05X SSC [solution 2]).  Slides were dried by centrifugation at 1000 rpm for 2 minutes and scanned within an hour of washing.  Fluorescence intensity was measured with Axon GenePix 4000A or 4000B scanners and the data was captured with GenePix 4.0 software. Following exclusion of suboptimal spots, intensity ratios were constructed by subtracting local background from foreground for each spot and normalizing one channel so that the mean foreground fluorescence of both channels across the slide was equal.

source_name: Zimbabwe30 adult males

source_name: St Louis adult males


title: ED1 18BPFCy3 vs PFCy5 600630GEO


title: E78a

source_name: Hikone R adult males

source_name: Zimbabwe29 adult males


title: WD3 18BPFCy3 vs PFCy5 740680GEO

source_name: 635nm (Cy5) channel, wing disc at pupariation

source_name: 532nm (Cy3) channel, wing disc at approximately 18 hours before pupariation


title: SG2 18BPFCy3 vs PFCy5 710650GEO

source_name: 635nm (Cy5) channel, salivary gland at pupariation

source_name: 532nm (Cy3) channel, salivary gland at approximately 18 hours before pupariation


title: E78b

source_name: Oregon R adult males

source_name: Zimbabwe2 adult males


title: FKS1(5251) #1

status: Public on May 30 2003

first: Garrett

middle: M

last: Frampton

address: 9 Cambridge Center

department: Biology

institute: Whitehead Instute - MIT

description:
The fks1 mutant strain (5251) was grown overnight in YPD medium, diluted 1:50 and grown to the mid-log phase (OD600 1.6-1.8) and harvested by centrifugation. Total RNA was isolated by a hot phenol procedure (Schmitt et al. 1990, Nucleic Acids Research 18: 3091-3092). Target probe preparation was done according to Affymetrix Gene Expression Technical Manual (Affymetrix, Santa Clara CA). Briefly, first strand and double-stranded cDNA were synthesized from total RNA (10-40 ug) (SuperScript II RT). cDNA was converted to biotinylated complementary RNA by in vitro transcription containing T7 RNA polymerase (Enzo Biochem). The cRNA product was cleaned up on RNeasy spin columns (Qiagen) followed by quantitation spectrophotometrically (UV lambda=260 nm) and by gel electrophoresis. About 20 ug of cRNA was fragmented to yield 35-200 bases fragments before hybridization. RNA probes were hybridized to the entire yeast genome microarray (YG-S98, Affymetrix) for 16 hours at 45 C. After washing in a non-stringent and stringent wash buffer, the probe arrays were stained with streptavidin phycoerythrin in the GeneChip Fluidics Station and scanned with the Affymetrix GeneChip Scanner according to manufacturer’s instructions. Gene chips were analyzed and normalized by GeneChip Analysis Suite.
Keywords = saccharomyces chitin glucosamine

organism: Saccharomyces cerevisiae

source_name: Total RNA


title: ED3 18BPFCy3 vs PFCy5 630670GEO


title: Drosophila ADAR Targets S1_N=2

status: Public on Jul 14 2003

city: Baltimore

first: Shuli

last: Xia

state: MD

institute: Kennedy-Krieger Institute

description: The aim of this experiment is to identify new targets of Drosophila adenosine deaminase acting on RNA (dADAR) by enriching Inosine-containing mRNA (I-mRNA) from wild type (C-S) and dADAR mutant flies and then hybridization with Drosophila cDNA arrays. mRNA was isolated from fly heads using Fasttrack mRNA isolation kit (Invitrogen, Carlsbad, California) via manufacturer’s instruction. Inosine-containing mRNA was isolated with the anti-inosine affinity column. 50 mg mRNA from wild type or dADAR mutant flies was heated to 70°C for 3 min, quickly put on ice for 5 min, then subjected on 1 ml of anti-inosine protein A-Sepharose column respectively. The mRNA was allowed to pass the column three times by gravity force. The column was then washed with 20 ml binding buffer (PBS plus 1 mM ATP, 0.1mM GTP, 1 mM CTP, 1 mM UTP, 5 mM EDTA). The bound mRNA was eluted with 2 ml binding buffer containing 1 mM ITP. The eluted fraction (2 ml) was collected. In vitro amplifying of mRNA was performed according to the method developed by Bough et al. RNA (3.5 ug) was labeled with Cy3 (C-S) or Cy5 (dADAR-/-) according to the method of Bough et al. Hybridazation was carried on at 64°C for 16-18 h. The next day, the slide was washed in a series of SDS/SSC before scanning. The data collected from the scanning were analyzed using GenePix Pro 3.0 (Axon Instruments, Union City, CA).

source_name: I-mRNA from Canton-S wild type, labeled with Cy3

source_name: I-mRNA from dADAR-/-, labeled with Cy5


title: WT (BY4741) +GlcN (SS) #3

description:
Wild type parental strain (BY4741) cells grown in glucosamine (15 mM) overnight were diluted 1:50 into fresh YPD supplemented with glucosamine and allowed to grow to a mid-log phase (OD600 1.6-1.8), and harvested by centrifugation. Total RNA was isolated by a hot phenol procedure (Schmitt et al. 1990, Nucleic Acids Research 18: 3091-3092). Target probe preparation was done according to Affymetrix Gene Expression Technical Manual (Affymetrix, Santa Clara CA). Briefly, first strand and double-stranded cDNA were synthesized from total RNA (10-40 ug) (SuperScript II RT). cDNA was converted to biotinylated complementary RNA by in vitro transcription containing T7 RNA polymerase (Enzo Biochem). The cRNA product was cleaned up on RNeasy spin columns (Qiagen) followed by quantitation spectrophotometrically (UV lambda=260 nm) and by gel electrophoresis. About 20 ug of cRNA was fragmented to yield 35-200 bases fragments before hybridization. RNA probes were hybridized to the entire yeast genome microarray (YG-S98, Affymetrix) for 16 hours at 45 C. After washing in a non-stringent and stringent wash buffer, the probe arrays were stained with streptavidin phycoerythrin in the GeneChip Fluidics Station and scanned with the Affymetrix GeneChip Scanner according to manufacturer’s instructions. Gene chips were analyzed and normalized by GeneChip Analysis Suite.  Three replicates labeled #1, #2, and #3 were performed.
Keywords = saccharomyces chitin glucosamine


title: MG2 18BPFCy3 vs PFCy5 710570GEO

source_name: 635nm (Cy5) channel, midgut at pupariation

source_name: 532nm (Cy3) channel, midgut at approximately 18 hours before pupariation


title: WD1 18BPFCy3 vs PFCy5 600630GEO

source_name: 635nm (Cy5) channel, wing disc at pupariation, one of two hybridizations using sample No. 1

source_name: 532nm (Cy3) channel, wing disc at approximately 18 hours before pupariation, one of two hybridizations using sample No. 1


title: WT (BY4741) +GlcN (SS) #2


title: SAGE2_20hrNo2Cy5 vs ControlNo2 Cy3_550460GEO

description:
The MIAME Checklist
Experiment Design:
·	Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or is it designed to study the effects of a gene knock-out?
Comparisons between the salivary glands upon the RNAi of sage gene vs. the control.  Total RNA samples were amplified according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.  Samples from the salivary glands after the RNAi of sage gene were hybridized against the control samples on each microarray.
·	Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
Each array tested the change of gene expression in salivary glands upon the RNAi of sage gene.
·	The number of hybridizations performed in the experiment.
Three replicate hybridizations using independent samples.
·	The type of reference used for the hybridizations, if any.
N/A.
·	Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
See above.
·	Quality control steps taken: for example, replicates or dye swaps.
Dye swaps were used.  Three replicates in total.
·	URL of any supplemental websites or database accession numbers
Samples used, extract preparation and labeling:
·	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.  See above.  Tissues/Organs were dissected from 9 or 10 individuals for each sample.
·	Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.  Animals were grown on standard corn meal medium with 0.05% bromophenol blue at 25 degrees Celsius.
·	Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol
Total RNA was extracted using TriZOL.  2 ug of total RNA from each sample was amplified with T7 RNA polymerase using a one-round linear amplification protocol.  RNA extraction and amplification were according to Baugh, L. R., Hill, A. A., Brown, E. L., and Hunter, C. P. (2001). Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29, E29.
·	Labeling protocol(s).
Priming step with random hexamers.  Labeling step using Superscript II, buffer, DTT, dUTP-Cy3, dUTP-Cy5 and free nucleotides.  Cleanup using Qiagen PCR cleanup kit.  Hybridization to slide in a TE, SSC, SDS solution with poly-A as a blocker.  Hybridization chambers from DIE-TECH, incubated for 10 minutes at 42 degrees followed by approximately 15-hour incubation at 64 degrees.  Three washes with successively more dilute solutions of SSC, including SDS in the first wash, then a centrifuge spin to dry.  Arrays were stored in the dark until scanned.  See K.P. White and K.C. Burtis “Drosophila microarrays: from arrayer construction to hybridization” in Drosophila Protocols, Cold Spring Harbor Press, 2000 for microarray protocols.
·	External controls (spikes).
Hybridization procedures and parameters:
·	The protocol and conditions used during hybridization, blocking and washing.
See above.
Measurement data and specifications:
·	The quantitations based on the images.
Images were gridded and spots were quantified with GenePix software (Axon Instruments)
·	The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following:
o	Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
Slides were scanned on an Axon 4000B scanner using Genepix Pro 3.0 software.
o	Type of image analysis software used: specifications should be stated in the materials and methods.
The program GenePix Pro 3.0 from Axon Instruments was used to analyse the data.
o	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
(F635 Median - B635), (F532 Median - B532), and (Ratio of Medians) were the raw measurements produced by the software.  The detailed information for these measurements is available at http://www.axon.com/manuals/GenePix_Pro_4.0_User_Guide_Rev_E.pdf
o	The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
See *.xls files attached.
o	Data selection and transformation procedures.
Data for all spots are filtered.  For a spot to pass quality control, 3 conditions need to be met: (1). There is no automatic or manual flag during image gridding using GenePix Pro software.  (2). For this spot, at least 1 channel is of at least 75% of the pixels are one SD above background.  (3). The net signal noise ratio should be at least 1 for at least one channel.  For the channel 635 nm wavelength, the signal noise ratio is the ratio between (F635_median-B635_median) to the background median.  The background median is the bigger of the median of B635-median of all the spots on the whole array, and B-median of this spot.  For the channel 532 nm wavelength, F532_median and B532_median are used.  Ratios between net signals are log2 transformed and analyzed as detailed in the paper.
o	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
See the web tables attached for the paper.  Additionally, see the column for Passed and normalized ratio of median in the *.xls files attached.  Data before and after data selection is in one file.
Array Design:
·	General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).
Spotted array using PCR fragments of approximately 95% of the genes in the D. melanogaster BDGP genome.  Slides were coated with polylysine, printed using a GeneMachines OmniGrid printer with DNA suspended in 3X SSC, and postprocessed using the DCE protocol of Diehl, F., Grahlmann, S., Beier, M. & Hoheisel, J.D. “Manufacturing DNA microarrays of high spot homogeneity and reduced background signal.” Nucleic Acids Research 29, e38 (2001).
·	For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given.
The KP numbers identify these and can be tied to the data files.  The ID of the respective reporter is included in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
·	For each reporter, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
The FBGN and CG numbers, primer pairs, sequences, and KP number (to tie them to the spots on the array) are all listed in the Excel file Web Table PCR Primer and Amplicon Sequence.xls.
·	For commercial arrays: a reference to the manufacturer should be provided, including a catalogue number and references to the manufacturer’s website if available.
·	For non-commercial arrays, the following details should be provided:
o	The source of the reporter molecules: for example, the cDNA or oligo collection used, with references.
o	The method of reporter preparation.
o	The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking.
o	Any ad

source_name: 635nm (Cy5) channel, salivary gland upon the RNAi of sage gene

source_name: 532nm (Cy3) channel, control


title: SAGE2_20hrNo1Cy3 vs ControlNo1 Cy5_560440GEO

source_name: 635nm (Cy5) channel, control

source_name: 532nm (Cy3) channel, salivary gland upon the RNAi of sage gene


title: MG1 18BPFCy3 vs PFCy5 780620GEO


title: CNS4 18BPFCy3 vs PFCy5 600600GEO

source_name: 635nm (Cy5) channel, central nervous system at pupariation

source_name: 532nm (Cy3) channel, central nervous system at approximately 18 hours before pupariation


title: WT (BY4741)+GlcN (2hr) #1

description:
An overnight culture of parental strain wild type (BY4741) grown in YPD was diluted 50-fold in fresh YPD, grown on an Orbital shaker for 2 or 3 hours, then glucosamine was added to a final concentration of 15 mM. Cells were grown in glucosamine for 2 hours to an OD600 of 1.6-1.8 and harvested by centrifugation. Total RNA was isolated by a hot phenol procedure (Schmitt et al. 1990, Nucleic Acids Research 18: 3091-3092). Target probe preparation was done according to Affymetrix Gene Expression Technical Manual (Affymetrix, Santa Clara CA). Briefly, first strand and double-stranded cDNA were synthesized from total RNA (10-40 ug) (SuperScript II RT). cDNA was converted to biotinylated complementary RNA by in vitro transcription containing T7 RNA polymerase (Enzo Biochem). The cRNA product was cleaned up on RNeasy spin columns (Qiagen) followed by quantitation spectrophotometrically (UV lambda=260 nm) and by gel electrophoresis. About 20 ug of cRNA was fragmented to yield 35-200 bases fragments before hybridization. RNA probes were hybridized to the entire yeast genome microarray (YG-S98, Affymetrix) for 16 hours at 45 C. After washing in a non-stringent and stringent wash buffer, the probe arrays were stained with streptavidin phycoerythrin in the GeneChip Fluidics Station and scanned with the Affymetrix GeneChip Scanner according to manufacturer’s instructions. Gene chips were analyzed and normalized by GeneChip Analysis Suite.  Three replicates labeled #1, #2, and #3 were performed.
Keywords = saccharomyces chitin glucosamine


title: SG1 18BPFCy3 vs PFCy5 640630GEO

source_name: 635nm (Cy5) channel, salivary gland at pupariation


title: WD2 18BPFCy3 vs PFCy5 600630GEO

source_name: 635nm (Cy5) channel, wing disc at pupariation

source_name: 532nm (Cy3) channel, wing disc at approximately 18 hours before pupariation


title: WT (BY4741)+GlcN (2hr) #2


title: SAGE2_20hrNo4Cy5 vs ControlNo4 Cy3_560460GEO


title: WT (BY4741) #3

description:
An overnight culture of parental wild type strain (BY4741) grown in YPD was diluted 50-fold in fresh YPD, grown further to an OD_600 of 1.6-1.8 and harvested by centrifugation. Total RNA was isolated by a hot phenol procedure (Schmitt et al. 1990, Nucleic Acids Research 18: 3091-3092). Target probe preparation was done according to Affymetrix Gene Expression Technical Manual (Affymetrix, Santa Clara CA). Briefly, first strand and double-stranded cDNA were synthesized from total RNA (10-40 ug) (SuperScript II RT). cDNA was converted to biotinylated complementary RNA by in vitro transcription containing T7 RNA polymerase (Enzo Biochem). The cRNA product was cleaned up on RNeasy spin columns (Qiagen) followed by quantitation spectrophotometrically (UV lambda = 260 nm) and by gel electrophoresis. About 20 ug of cRNA was fragmented to yield 35-200 bases fragments before hybridization. RNA probes were hybridized to the entire yeast genome microarray (YG-S98, Affymetrix) for 16 hours at 45 C. After washing in a non-stringent and stringent wash buffer, the probe arrays were stained with streptavidin phycoerythrin in the GeneChip Fluidics Station and scanned with the Affymetrix GeneChip Scanner according to manufacturer’s instructions. Gene chips were analyzed and normalized by GeneChip Analysis Suite. Three replicates labeled #1, #2, and #3 were performed.
Keywords = saccharomyces chitin glucosamine


title: Drosophila ADAR Targets S2_N=2


title: WT (BY4741)+GlcN (2hr) #3

description: An overnight culture of parental strain wild type (BY4741) grown in YPD was diluted 50-fold in fresh YPD, grown on an Orbital shaker for 2 or 3 hours, then glucosamine was added to a final concentration of 15 mM. Cells were grown in glucosamine for 2 hours to an OD600 of 1.6-1.8 and harvested by centrifugation. Total RNA was isolated by a hot phenol procedure (Schmitt et al. 1990, Nucleic Acids Research 18: 3091-3092). Target probe preparation was done according to Affymetrix Gene Expression Technical Manual (Affymetrix, Santa Clara CA). Briefly, first strand and double-stranded cDNA were synthesized from total RNA (10-40 ug) (SuperScript II RT). cDNA was converted to biotinylated complementary RNA by in vitro transcription containing T7 RNA polymerase (Enzo Biochem). The cRNA product was cleaned up on RNeasy spin columns (Qiagen) followed by quantitation spectrophotometrically (UV lambda=260 nm) and by gel electrophoresis. About 20 ug of cRNA was fragmented to yield 35-200 bases fragments before hybridization. RNA probes were hybridized to the entire yeast genome microarray (YG-S98, Affymetrix) for 16 hours at 45 C. After washing in a non-stringent and stringent wash buffer, the probe arrays were stained with streptavidin phycoerythrin in the GeneChip Fluidics Station and scanned with the Affymetrix GeneChip Scanner according to manufacturer’s instructions. Gene chips were analyzed and normalized by GeneChip Analysis Suite.  Three replicates labeled #1, #2, and #3 were performed.


title: 269800AB_4

status: Public on May 27 2003

city: New York

first: Elisa

last: Wurmbach

state: NY

address: 1 Gustave L. Levy Place

department: Hematology/Oncology

institute: Mount Sinai School of Medicine

description:
To investigate the gene program activated by serotonergic hallucinogens in mouse somatosensory cortex, the effects of the 5-HT2A agonist (±)2,5-dimethoxy 4-iodoamphetamine (DOI) on gene expression was studied. The 5-HT2A serotonin receptor is a member of the 5-HT2 receptor subfamily of G protein–coupled receptors. The 5-HT2A receptor is thought to modulate processes related to cognition, appetite, mood, anxiety, sleep, and sexual behavior. Abnormal function of this receptor has been implicated in neuropsychiatric disorders such as major depression and schizophrenia. In particular, the 5-HT2A receptor has been implicated in the modulation of the mechanisms related to psychosis. We compared gene response patterns in the somatosensory cortical brain area, which has a high density of 5-HT2A receptors and has been implicated in the action of hallucinogenic drugs. Mice (strain 129Sv) were injected with DOI (2 mg/kg or 10 mg/kg) or vehicle, animals were sacrificed after 1 h, and somatosensory cortex samples dissected on ice for RNA extraction. Seven independent microarray hybridizations from seven different DOI- or vehicle-treated mouse pairs were performed.

In this particular hybridization, 20 µg of RNA was used.
Keywords = somatosensory cortex 5-HT2A receptor DOI

source_name: Mouse somatosensory cortex 1 hour after 2 mg/kg DOI treatment

source_name: Mouse somatosensory cortex 1 hour after saline treatment


title: WT (BY4741) #2

description:
An overnight culture of parental wild type strain (BY4741) grown in YPD was diluted 50-fold in fresh YPD, grown further to an OD_600 of 1.6-1.8 and harvested by centrifugation. Total RNA was isolated by a hot phenol procedure (Schmitt et al. 1990, Nucleic Acids Research 18: 3091-3092). Target probe preparation was done according to Affymetrix Gene Expression Technical Manual (Affymetrix, Santa Clara CA). Briefly, first strand and double-stranded cDNA were synthesized from total RNA (10-40 ug) (SuperScript II RT). cDNA was converted to biotinylated complementary RNA by in vitro transcription containing T7 RNA polymerase (Enzo Biochem). The cRNA product was cleaned up on RNeasy spin columns (Qiagen) followed by quantitation spectrophotometrically (UV lambda = 260 nm) and by gel electrophoresis. About 20 ug of cRNA was fragmented to yield 35-200 bases fragments before hybridization. RNA probes were hybridized to the entire yeast genome microarray (YG-S98, Affymetrix) for 16 hours at 45 C. After washing in a non-stringent and stringent wash buffer, the probe arrays were stained with streptavidin phycoerythrin in the GeneChip Fluidics Station and scanned with the Affymetrix GeneChip Scanner according to manufacturer’s instructions. Gene chips were analyzed and normalized by GeneChip Analysis Suite.  Three replicates labeled #1, #2, and #3 were performed.
Keywords = saccharomyces chitin glucosamine

source_name: Total RNA


title: 225636DE_4

description:
To investigate the gene program activated by serotonergic hallucinogens in mouse somatosensory cortex, the effects of the 5-HT2A agonist (±)2,5-dimethoxy 4-iodoamphetamine (DOI) on gene expression was studied. The 5-HT2A serotonin receptor is a member of the 5-HT2 receptor subfamily of G protein–coupled receptors. The 5-HT2A receptor is thought to modulate processes related to cognition, appetite, mood, anxiety, sleep, and sexual behavior. Abnormal function of this receptor has been implicated in neuropsychiatric disorders such as major depression and schizophrenia. In particular, the 5-HT2A receptor has been implicated in the modulation of the mechanisms related to psychosis. We compared gene response patterns in the somatosensory cortical brain area, which has a high density of 5-HT2A receptors and has been implicated in the action of hallucinogenic drugs. Mice (strain 129Sv) were injected with DOI (2 mg/kg or 10 mg/kg) or vehicle, animals were sacrificed after 1 h, and somatosensory cortex samples dissected on ice for RNA extraction. Seven independent microarray hybridizations from seven different DOI- or vehicle-treated mouse pairs were performed.

In this particular hybridization, 15 µg of RNA was used.
Keywords = somatosensory cortex 5-HT2A receptor DOI


title: 269813CD_4

source_name: Mouse somatosensory cortex 1 hour after 10 mg/kg DOI treatment


title: 269802DC_4


title: ADR018

status: Public on Mar 26 2003

city: Evry

first: Jerome

last: Lamartine

address: 2 rue Gaston Cremieux

department: Departement de Radiobiologie et Radiopathologie

country: France

institute: CEA

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

Keywords = Human keratinocytes, gamma irradiation

organism: Homo sapiens

source_name: HaCaT keratinocytes 0 Gy 24h (reference)

source_name: HaCaT keratinocytes 0.5 Gy 24h


title: ADR003

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

Keywords = Human keratinocytes, gamma irradiation

source_name: HaCaT keratinocytes 2 Gy 24h


title: ADQ045

status: Public on Mar 25 2003

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

Keywords = human keratinocytes, gamma irradiation

source_name: HaCaT keratinocytes 0 Gy 24h (Reference)


title: ADQ044


title: ADQ018

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

Keywords = human keratinocytes, gamma irradiation

source_name: HaCaT keratinocytes 0 Gy (Reference)

source_name: HaCaT keratinocytes 0.5 Gy


title: ADQ017

status: Public on Mar 21 2003


title: 225635HG_4


title: ADR048


title: B(Sg) Heat SigmaSlide10

status: Public on Mar 14 2003

city: Tucson

first: Jennifer

last: Wilcox

state: AZ

department: EEB

institute: University of Arizona

description:
Experimental conditions imposed on symbionts within intact aphid hosts feeding on barley host plant. Reference sample was derived from feeding, non-stressed aphid/symbiont tissues at 19°C (normal culture conditions). Heat stress was imposed gradually from 19°C to 36°C over 2 hours.
Keywords = aphid endosymbiont

organism: Buchnera aphidicola (Schizaphis graminum)

source_name: Heat shock total RNA sample

source_name: Reference total RNA sample


title: B(Sg) Heat SigmaSlide9


title: ARD017


title: ADR008


title: ACX023

source_name: HaCaT keratinocytes 0.5 Gy 24 h


title: B(Sg) Heat SigmaSlide1


title: B(Sg) Heat SigmaSlide6


title: ACX020

status: Public on Mar 20 2003

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

Keywords = human keratinocytes, gamma irradiation

source_name: HaCaT Keratinocytes 0.5 Gy 24h

source_name: HaCaT Keratinocytes 0 Gy 24h (reference)


title: ACF013

status: Public on Mar 13 2003

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

Keywords = Human keratinocytes, gamma irradiation

source_name: HaCaT keratinocytes 0 Gy 3h (Reference)

source_name: HaCaT keratinocytes 2 Gy 3h


title: ACF012;ACF012

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

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

Keywords = Human keratinocytes, gamma radiations

source_name: HaCaT keratinocytes 2 Gy 3h;HaCaT keratinocytes 2 Gy 3h

source_name: HaCaT keratinocytes 0 Gy 3h (Reference);HaCaT keratinocytes 0 Gy 3h (Reference)


title: ADQ021

status: Public on Mar 11 2003

description:
The keratinocyte cell line HaCaT culture was performed in DMEM (Invitrogen), 10% FCS (Hyclone) at 37°C and 5% CO2 incubator. Cells were seeded at 10 000 cells/cm2 and cultured for three days (proliferation) or for ten days (differentiation). Medium was changed every two days.
Total RNA was isolated using the Trizol reagent (Invitrogen) and dissolved in RNAse-free water. RNA quality was checked by 1 % agarose gel electrophoresis and concentration was measured by absorbance at 260 nm.
Target cDNA was generated by direct incorporation of fluorescent nucleotide analogues during reverse transcription. For each labelling experiment, 20 µg of total RNA was submitted to an oligo(dT) primed reaction in the presence of 60 µM Cy3 or Cy5-dUTP (Amersham Biosciences), 500 µM each dATP, dCTP, dGTP, 200 µM dTTP, 4 µg oligo(dT) and 400 U of Superscript II (Invitrogen) in a final volume of 30 µl. After incubation at 42°C for 2 hours in a dark room, RNA was treated with 2 units of DNAse-free RNAse (Invitrogen) at 37°C for 15 min. The volume was adjusted to 500 µl with TE (10 mM TrisHCl pH 7.4, 1 mM EDTA) and non-incorporated nucleotides were separated from labelled cDNA by centrifugation in a YM-30 Microcon filter (Millipore Amicon). The volume of each labelled cDNA was then adjusted to 40 µl.
Cy3 and Cy5-labeled cDNAs were subsequently mixed with 10 µg of human Cot-1 DNA (Invitrogen), 10µg poly(A) (Sigma-Aldrich) and 10µg of yeast tRNA (Invitrogen) then precipitated with 0.5 volume of 7.5 M sodium acetate (pH 5.2) in ethanol. The pellet was dissolved in 15 µl of hybridisation solution (50% formamide, 1,75x Denhardt’s, 0,35% SDS and 6x SSPE). The probe was heated for 2 min at 100°C, incubated at 37°C for 20-30 min and placed between slide and coverslip. The arrays were incubated overnight at 42°C in an humidified slide chamber (Corning) and washed in a first bath of 0.1x SSC with 0.1% SDS for 15 minutes followed by two washing steps in 0.1x SSC for 15 min each at room temperature. The arrays were dried by centrifugation at 600 rpm for 6 min.
Florescence intensities of Cy3 (532 nm) and Cy5 (635 nm) were measured separately with a laser scanner (Axon Genepix4000B).
Keywords = HUman keratinocytes, gamma irradiation

source_name: HaCaT keratinocytes 0 Gy 3h

source_name: HaCaT keratinocytes 0.5 Gy 3h


title: ACX041

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

Keywords = human keratinocytes, gamma irradiation

source_name: HaCaT keratinocytes 0 Gy 3h (reference)


title: ACF017

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

Keywords = Human keratinocytes, gamma irradiation


title: ACF021

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

Keywords = Human keratinocytes, gamma irradiation

source_name: HaCaT keratinocytes 0.5Gy 3h


title: ADQ020

description:
The keratinocyte cell line HaCaT culture was performed in DMEM (Invitrogen), 10% FCS (Hyclone) at 37°C and 5% CO2 incubator. Cells were seeded at 10 000 cells/cm2 and cultured for three days (proliferation) or for ten days (differentiation). Medium was changed every two days.
Total RNA was isolated using the Trizol reagent (Invitrogen) and dissolved in RNAse-free water. RNA quality was checked by 1 % agarose gel electrophoresis and concentration was measured by absorbance at 260 nm.
Target cDNA was generated by direct incorporation of fluorescent nucleotide analogues during reverse transcription. For each labelling experiment, 20 µg of total RNA was submitted to an oligo(dT) primed reaction in the presence of 60 µM Cy3 or Cy5-dUTP (Amersham Biosciences), 500 µM each dATP, dCTP, dGTP, 200 µM dTTP, 4 µg oligo(dT) and 400 U of Superscript II (Invitrogen) in a final volume of 30 µl. After incubation at 42°C for 2 hours in a dark room, RNA was treated with 2 units of DNAse-free RNAse (Invitrogen) at 37°C for 15 min. The volume was adjusted to 500 µl with TE (10 mM TrisHCl pH 7.4, 1 mM EDTA) and non-incorporated nucleotides were separated from labelled cDNA by centrifugation in a YM-30 Microcon filter (Millipore Amicon). The volume of each labelled cDNA was then adjusted to 40 µl.
Cy3 and Cy5-labeled cDNAs were subsequently mixed with 10 µg of human Cot-1 DNA (Invitrogen), 10µg poly(A) (Sigma-Aldrich) and 10µg of yeast tRNA (Invitrogen) then precipitated with 0.5 volume of 7.5 M sodium acetate (pH 5.2) in ethanol. The pellet was dissolved in 15 µl of hybridisation solution (50% formamide, 1,75x Denhardt’s, 0,35% SDS and 6x SSPE). The probe was heated for 2 min at 100°C, incubated at 37°C for 20-30 min and placed between slide and coverslip. The arrays were incubated overnight at 42°C in an humidified slide chamber (Corning) and washed in a first bath of 0.1x SSC with 0.1% SDS for 15 minutes followed by two washing steps in 0.1x SSC for 15 min each at room temperature. The arrays were dried by centrifugation at 600 rpm for 6 min.
Florescence intensities of Cy3 (532 nm) and Cy5 (635 nm) were measured separately with a laser scanner (Axon Genepix4000B).
Keywords = human keratinocytes, gamma irradiation


title: ADQ048

status: Public on Mar 10 2003

description:
The keratinocyte cell line HaCaT culture was performed in DMEM (Invitrogen), 10% FCS (Hyclone) at 37°C and 5% CO2 incubator. Cells were seeded at 10 000 cells/cm2 and cultured for three days (proliferation) or for ten days (differentiation). Medium was changed every two days.
Total RNA was isolated using the Trizol reagent (Invitrogen) and dissolved in RNAse-free water. RNA quality was checked by 1 % agarose gel electrophoresis and concentration was measured by absorbance at 260 nm.
Target cDNA was generated by direct incorporation of fluorescent nucleotide analogues during reverse transcription. For each labelling experiment, 20 µg of total RNA was submitted to an oligo(dT) primed reaction in the presence of 60 µM Cy3 or Cy5-dUTP (Amersham Biosciences), 500 µM each dATP, dCTP, dGTP, 200 µM dTTP, 4 µg oligo(dT) and 400 U of Superscript II (Invitrogen) in a final volume of 30 µl. After incubation at 42°C for 2 hours in a dark room, RNA was treated with 2 units of DNAse-free RNAse (Invitrogen) at 37°C for 15 min. The volume was adjusted to 500 µl with TE (10 mM TrisHCl pH 7.4, 1 mM EDTA) and non-incorporated nucleotides were separated from labelled cDNA by centrifugation in a YM-30 Microcon filter (Millipore Amicon). The volume of each labelled cDNA was then adjusted to 40 µl.
Cy3 and Cy5-labeled cDNAs were subsequently mixed with 10 µg of human Cot-1 DNA (Invitrogen), 10µg poly(A) (Sigma-Aldrich) and 10µg of yeast tRNA (Invitrogen) then precipitated with 0.5 volume of 7.5 M sodium acetate (pH 5.2) in ethanol. The pellet was dissolved in 15 µl of hybridisation solution (50% formamide, 1,75x Denhardt’s, 0,35% SDS and 6x SSPE). The probe was heated for 2 min at 100°C, incubated at 37°C for 20-30 min and placed between slide and coverslip. The arrays were incubated overnight at 42°C in an humidified slide chamber (Corning) and washed in a first bath of 0.1x SSC with 0.1% SDS for 15 minutes followed by two washing steps in 0.1x SSC for 15 min each at room temperature. The arrays were dried by centrifugation at 600 rpm for 6 min.
Florescence intensities of Cy3 (532 nm) and Cy5 (635 nm) were measured separately with a laser scanner (Axon Genepix4000B).
Keywords = Human keratinocytes, differentiation program

source_name: HaCaT keratinocytes day10 cell density differentiated

source_name: HaCaT keratinocytes day3 proliferating


title: ACG010

description:
The keratinocyte cell line HaCaT culture was performed in DMEM (Invitrogen), 10% FCS (Hyclone) at 37°C and 5% CO2 incubator. Cells were seeded at 10 000 cells/cm2 and cultured for three days (proliferation) or for ten days (differentiation). Medium was changed every two days.
Total RNA was isolated using the Trizol reagent (Invitrogen) and dissolved in RNAse-free water. RNA quality was checked by 1 % agarose gel electrophoresis and concentration was measured by absorbance at 260 nm.
Target cDNA was generated by direct incorporation of fluorescent nucleotide analogues during reverse transcription. For each labelling experiment, 20 µg of total RNA was submitted to an oligo(dT) primed reaction in the presence of 60 µM Cy3 or Cy5-dUTP (Amersham Biosciences), 500 µM each dATP, dCTP, dGTP, 200 µM dTTP, 4 µg oligo(dT) and 400 U of Superscript II (Invitrogen) in a final volume of 30 µl. After incubation at 42°C for 2 hours in a dark room, RNA was treated with 2 units of DNAse-free RNAse (Invitrogen) at 37°C for 15 min. The volume was adjusted to 500 µl with TE (10 mM TrisHCl pH 7.4, 1 mM EDTA) and non-incorporated nucleotides were separated from labelled cDNA by centrifugation in a YM-30 Microcon filter (Millipore Amicon). The volume of each labelled cDNA was then adjusted to 40 µl.
Cy3 and Cy5-labeled cDNAs were subsequently mixed with 10 µg of human Cot-1 DNA (Invitrogen), 10µg poly(A) (Sigma-Aldrich) and 10µg of yeast tRNA (Invitrogen) then precipitated with 0.5 volume of 7.5 M sodium acetate (pH 5.2) in ethanol. The pellet was dissolved in 15 µl of hybridisation solution (50% formamide, 1,75x Denhardt’s, 0,35% SDS and 6x SSPE). The probe was heated for 2 min at 100°C, incubated at 37°C for 20-30 min and placed between slide and coverslip. The arrays were incubated overnight at 42°C in an humidified slide chamber (Corning) and washed in a first bath of 0.1x SSC with 0.1% SDS for 15 minutes followed by two washing steps in 0.1x SSC for 15 min each at room temperature. The arrays were dried by centrifugation at 600 rpm for 6 min.
Florescence intensities of Cy3 (532 nm) and Cy5 (635 nm) were measured separately with a laser scanner (Axon Genepix4000B).


title: ACX031

status: Public on Mar 07 2003

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

source_name: HaCat keratinocytes 2Gy 3h

source_name: HaCat keratinocytes 0 Gy 3h (Reference)


title: ACG006

description:
The keratinocyte cell line HaCaT culture was performed in DMEM (Invitrogen), 10% FCS (Hyclone) at 37°C and 5% CO2 incubator. Cells were seeded at 10 000 cells/cm2 and cultured for three days (proliferation) or for ten days (differentiation). Medium was changed every two days. The cell cycle analysis was performed on two independent experiments at each experimental point.
Total RNA was isolated using the Trizol reagent (Invitrogen) and dissolved in RNAse-free water. RNA quality was checked by 1 % agarose gel electrophoresis and concentration was measured by absorbance at 260 nm.
Target cDNA was generated by direct incorporation of fluorescent nucleotide analogues during reverse transcription. For each labelling experiment, 20 µg of total RNA was submitted to an oligo(dT) primed reaction in the presence of 60 µM Cy3 or Cy5-dUTP (Amersham Biosciences), 500 µM each dATP, dCTP, dGTP, 200 µM dTTP, 4 µg oligo(dT) and 400 U of Superscript II (Invitrogen) in a final volume of 30 µl. After incubation at 42°C for 2 hours in a dark room, RNA was treated with  2 units of DNAse-free RNAse (Invitrogen) at 37°C for 15 min. The volume was adjusted to 500 µl with TE (10 mM TrisHCl pH 7.4, 1 mM EDTA) and non-incorporated nucleotides were separated from labelled cDNA by centrifugation in a YM-30 Microcon filter (Millipore Amicon). The volume of each labelled cDNA was then adjusted to 40 µl.
Cy3 and Cy5-labeled cDNAs were subsequently mixed with 10 µg of human Cot-1 DNA (Invitrogen), 10µg poly(A) (Sigma-Aldrich) and 10µg of yeast tRNA (Invitrogen) then precipitated with 0.5 volume of 7.5 M sodium acetate (pH 5.2) in ethanol. The pellet was dissolved in 15 µl of hybridisation solution (50% formamide, 1,75x Denhardt’s, 0,35% SDS and 6x SSPE). The probe was heated for 2 min at 100°C, incubated at 37°C for 20-30 min and placed between slide and coverslip. The arrays were incubated overnight at 42°C in an humidified slide chamber (Corning) and washed in a first bath of 0.1x SSC with 0.1% SDS for 15 minutes followed by two washing steps in 0.1x SSC for 15 min each at room temperature. The arrays were dried by centrifugation at 600 rpm for 6 min.
Fluorescence intensities of Cy3 (532 nm) and Cy5 (635 nm) were measured separately with a laser scanner (Axon Genepix4000B).
Keywords = Human keratinocytes, differentiation program

source_name: HaCat day10 cell-density differentiated

source_name: HaCat day3 proliferating


title: ACG005

description:
The keratinocyte cell line HaCaT culture was performed in DMEM (Invitrogen), 10% FCS (Hyclone) at 37°C and 5% CO2 incubator. Cells were seeded at 10 000 cells/cm2 and cultured for three days (proliferation) or for ten days (differentiation). Medium was changed every two days.
Total RNA was isolated using the Trizol reagent (Invitrogen) and dissolved in RNAse-free water. RNA quality was checked by 1 % agarose gel electrophoresis and concentration was measured by absorbance at 260 nm.
Target cDNA was generated by direct incorporation of fluorescent nucleotide analogues during reverse transcription. For each labelling experiment, 20 µg of total RNA was submitted to an oligo(dT) primed reaction in the presence of 60 µM Cy3 or Cy5-dUTP (Amersham Biosciences), 500 µM each dATP, dCTP, dGTP, 200 µM dTTP, 4 µg oligo(dT) and 400 U of Superscript II (Invitrogen) in a final volume of 30 µl. After incubation at 42°C for 2 hours in a dark room, RNA was treated with  2 units of DNAse-free RNAse (Invitrogen) at 37°C for 15 min. The volume was adjusted to 500 µl with TE (10 mM TrisHCl pH 7.4, 1 mM EDTA) and non-incorporated nucleotides were separated from labelled cDNA by centrifugation in a YM-30 Microcon filter (Millipore Amicon). The volume of each labelled cDNA was then adjusted to 40 µl.
Cy3 and Cy5-labeled cDNAs were subsequently mixed with 10 µg of human Cot-1 DNA (Invitrogen), 10µg poly(A) (Sigma-Aldrich) and 10µg of yeast tRNA (Invitrogen) then precipitated with 0.5 volume of 7.5 M sodium acetate (pH 5.2) in ethanol. The pellet was dissolved in 15 µl of hybridisation solution (50% formamide, 1,75x Denhardt’s, 0,35% SDS and 6x SSPE). The probe was heated for 2 min at 100°C, incubated at 37°C for 20-30 min and placed between slide and coverslip. The arrays were incubated overnight at 42°C in an humidified slide chamber (Corning) and washed in a first bath of 0.1x SSC with 0.1% SDS for 15 minutes followed by two washing steps in 0.1x SSC for 15 min each at room temperature. The arrays were dried by centrifugation at 600 rpm for 6 min.
Florescence intensities of Cy3 (532 nm) and Cy5 (635 nm) were measured separately with a laser scanner (Axon Genepix4000B).
Keywords = Human keratinocytes, differentiation program

source_name: HaCat keratinocytes day10 cell density differentiated

source_name: HaCat keratinocytes day3 proliferating