title: ACX017

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 = gamma irradiation, human keratinocytes


title: ACX027

status: Public on Mar 07 2003

type: RNA

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 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

molecule: total RNA

organism: Homo sapiens

source_name: HaCat keratinocytes 0 Gy 3h (Reference)

source_name: HaCat keratinocytes 2 Gy 3h


title: FKS1(5251) +GlcN (2hr)  #1

status: Public on May 30 2003

city: Cambridge

first: Garrett

middle: M

last: Frampton

state: MA

address: 9 Cambridge Center

department: Biology

country: USA

institute: Whitehead Instute - MIT

description:
The fks1 mutant strain (5251) was grown overnight in YPD medium, diluted 1:50, grown for 2 hrs and then supplemented with 15mM glucosamine. Cells were grown 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.

Keywords = saccharomyces chitin glucosamine

organism: Saccharomyces cerevisiae

source_name: Total RNA


title: mefloquine-treated NG108 neuronal cells 7

status: Public on Feb 27 2003

city: Silver Spring

first: Geoffrey

middle: S

last: Dow

state: MD

address: Robert Grant Ave.

department: Division of Experimental Therapeutics, Building 503

institute: Walter Reed Army Institute of Research

description:
NG108 rat neuronal cell culture exposed to 4400 ng/ml mefloquine (treated) for two hours.
10 µg total RNA hybridized per chip. RNA STAT 60 used to extract RNA.  Standard Affymetrix chip scaling factors used.  No other data transformation.

organism: Rattus norvegicus


title: ACX015

status: Public on Mar 06 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 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).

NormalizationFactor:RatioOfMedians=1.49613
NormalizationFactor:RatioOfMeans=1.42747
NormalizationFactor:MedianOfRatios=1.39915
NormalizationFactor:MeanOfRatios=1.44113
NormalizationFactor:RegressionRatio=0.860883
Keywords = human keratinocytes, gamma irradiation

source_name: HaCat keratinocytes 0 Gy 3h (reference)


title: mefloquine-treated NG108 neuronal cells 5

source_name: NG108 rat neuronal cell culture exposed to 4400 ng/ml mefloquine


title: ACX016


title: ACG025

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 day10 cell density differentiated

source_name: HaCaT day3 proliferating


title: mefloquine-treated NG108 neuronal cells 4


title: mefloquine-treated NG108 neuronal cells 8


title: mefloquine-treated NG108 neuronal cells 2


title: DMSO-control NG108 neuronal cells 8

description:
NG108 rat neuronal cell culture exposed to 0.25% DMSO (control) for two hours.
10 µg total RNA hybridized per chip. RNA STAT 60 used to extract RNA.  Standard Affymetrix chip scaling factors used.  No other data transformation.


title: mefloquine-treated NG108 neuronal cells 1


title: mefloquine-treated NG108 neuronal cells 6


title: ACG011

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 keratnocytes, differentiation program

source_name: HaCaT keratinocytes day3 proliferating

source_name: HaCaT keratinocytes day10 cell density differentiated


title: DMSO-control NG108 neuronal cells 7

source_name: NG108 rat neuronal cell culture exposed to 0.25% DMSO


title: DMSO-control NG108 neuronal cells 6


title: DMSO-control NG108 neuronal cells 1


title: DMSO-control NG108 neuronal cells 3


title: mefloquine-treated NG108 neuronal cells 3


title: DMSO-control NG108 neuronal cells 5


title: DMSO-control NG108 neuronal cells 4


title: Life cycle stage - gametocyte

status: Public on Dec 23 2002

type: protein

city: La Jolla

first: Laurence

last: Florens

state: CA

address: SR-11, 10550 North Torrey Pines Road

department: Department of Cell Biology

institute: The Scripps Research Institute

description:
A proteomic view of the gametocyte (sexual) stage.
Starting with synchronized asexual parasites grown in suspension culture, gametocytes were prepared by daily media changes of static cultures at 37°C. When there were very few mature asexual stages present, gametocyte-infected erythrocytes were collected from the 52.5%/45% and 45%/30% interfaces of a Percoll gradient. The gametocytes consisted mostly of stage IV and V parasites with minor contamination (<3%) from mixed asexual stage parasites.
Proteome was analyzed by multidimentional protein identification technology (MudPIT), which combines in-line, high-resolution liquid chromatography and tandem mass spectrometry.

molecule: protein

organism: Plasmodium falciparum 3D7

source_name: gametocytes


title: DMSO-control NG108 neuronal cells 2


title: human-macaque testis

status: Public on Nov 24 2002

city: Tokyo

first: Naoki

last: Osada

state: Tokyo

address: 1-23-1, Toyama-cho, Shinjuku-ku

country: Japan

institute: National Institute of Infectious Diseases

description: The cynomolgus monkey testicular RNA was obtained from only one 15-year-old male cynomolgus monkey, and human testiscular RNA was purchased from Clontech.  The RNA was isolated with Trizol (Life Technologies) and purified with Oligo-Tex (TakaraBio).  Both 0.7 µg mRNA probes were labeled with Cy3- and Cy5- dioxynucleotide (Pharmacia) and co-hybridized to DNA spots. After the hybridization and washing procedure, slides were scanned with ScanArray (GSI Inc.).

organism:
Macaca fascicularis
Homo sapiens

source_name: cynomolgus monkey testis

source_name: human testis


title: yra1-1TS_v_Yra1WT 051601 slide 1 scan 2

status: Public on Nov 25 2002

city: Boston

first: Haley

last: Hieronymus

address: 1 Jimmy Fund Way SM922

institute: Harvard Medical School and Dana-Farber Cancer Institute

description: Comparison of cDNA from RNA of yra1-1 temperature-sensitive mutant to RNA from Yra1 wildtype strain, both at the non-permissive temperature of 37°C

source_name: total RNA from yra1-1 temperature-sensitive cells at 37°C

source_name: total RNA from Yra1 wildtype cells at 37°C


title: yra1-1TS_v_Yra1WT 052801 slide 2 scan 3

description: Comparison of cDNA from RNA of yra1-1 temperature-sensitive mutant to RNA from Yra1 wildtype strain both at the non-permissive temperature of 37°C


title: yra1-1TS_v_Yra1WT 061401 slide 5b scan 4


title: 22-oct-2002-BrainAsubB-ab150

status: Public on Nov 11 2002

city: Lausanne

first: David

last: Vilanova

country: Switzerland

institute: Nestle Research Center

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.  After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hippocampal gene expression analysis. From these same 5 mice, the hippocampus was dissected from the mice according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), rinsed as described for livers, cut into 17-50 mg subsections, and frozen as described for livers.  Hippocampal lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Hippocampal tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hippocampal RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.

organism: Mus musculus

source_name: brain/hippocampus


title: mexyra1-1TS_v_Yra1WT 061401 slide 5a scan 5

description: Comparison of cDNA from RNA of yra1-1 temperature-sensitive mutant to RNA from Yra1 wildtype strain both at the non-permissive temperature of 37°C.


title: mex67-5TS_v_Mex67WT 061401 slide 2a scan 2

description: Yra1 binding level: Comparison of cDNA from RNA of mex67-5 temperature-sensitive mutant to RNA from Mex67 wildtype strain both at the non-permissive temperature of 37°C

source_name: total RNA from mex67-5 temperature-sensitive cells at 37°C

source_name: total RNA from Mex67 wildtype cells at 37°C


title: 22-oct-2002-liverF-Mu11KsubAsc

status: Public on Nov 05 2002

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.1% fungal oil and 1.9% fish oil. Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.
After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hepatic gene expression analysis. From these 5 mice, livers were dissected according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), which consisted of carefully dissecting out the liver, rinsing with 70% EtOH to remove adhering blood, trimming of adhering connective tissue with scissors, cutting into 100-150 mg subsections, freezing in liquid N2, and storing at -80°C until gene expression and lipid analysis. Hepatic lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Liver tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hepatic RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.

source_name: liver/hepatocytes


title: yra1-1TS_v_Yra1WT 051101 slide 3 scan 4


title: 22-oct-2002-BrainAsubA-ab150


title: yra1-1TS_v_Yra1WT 051101 slide 1 scan 4


title: 22-oct-2002-liverA1-Mu11KsubAsc

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.
After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hepatic gene expression analysis. From these 5 mice, livers were dissected according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), which consisted of carefully dissecting out the liver, rinsing with 70% EtOH to remove adhering blood, trimming of adhering connective tissue with scissors, cutting into 100-150 mg subsections, freezing in liquid N2, and storing at -80°C until gene expression and lipid analysis. Hepatic lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Liver tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hepatic RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.


title: mex67-5TS_v_Mex67WT 061101 slide 1 scan 7

description: Comparison of cDNA from RNA of mex67-5 temperature-sensitive mutant to RNA from Mex67 wildtype strain both at the non-permissive temperature of 37°C


title: mex67-5TS_v_Mex67WT 061401 slide 2b scan 3a


title: 22-oct-2002-liverD-Mu11KsubAsc

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.1% fungal oil (providing 0.5 dietary wt% AA and 1.0en%). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.
After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hepatic gene expression analysis. From these 5 mice, livers were dissected according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), which consisted of carefully dissecting out the liver, rinsing with 70% EtOH to remove adhering blood, trimming of adhering connective tissue with scissors, cutting into 100-150 mg subsections, freezing in liquid N2, and storing at -80°C until gene expression and lipid analysis. Hepatic lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Liver tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hepatic RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.


title: mex67-5TS_v_Mex67WT 061401 slide 3b scan 2


title: S805

city: Madison

first: Todd

last: Richmond

state: WI

address: One Science Court

department: Bioinformatics

institute: NimbleGen Systems Inc.

description:
Total mouse spleen RNA was obtained from Ambion, Inc. (Austin, TX). mRNA isolation was performed using oligo-dT hybrid capture on magnetic streptavidin beads using a commercially available kit (PolyATract System 1000, Promega, Madison, WI).  poly A+ RNA was converted to double-stranded cDNA using GibCo (Rockville, MD) BRL’s SuperScript Choice System and an oligo dT primer containing the T7 RNA polymerase promoter (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).  in vitro transcription (IVT) was used to produce biotin-labeled cRNA from the cDNA using the Ambion (Austin, TX) MEGAscript T7 kit. cRNA was purified using Qiagen RNeasy RNA purification columns  according to the manufacturer’s instructions. Before hybridization, cRNA was fragmented to an average size of 50-200 bp. Microarrays were hybridized with 12 ug cRNA in 300 ul, in the presence of 50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20 for 16 hours at 45 degrees C. After hybridization, arrays were washed in non-stringent (NS) buffer (6X SSPE, 0.01% (v/v) Tween 20) for 5 min at RT, followed by washing in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) for 30 min at 45 degrees C.  After washing, arrays were stained with streptavidin-Cy3 conjugate from Amersham Pharmacia for 25 min at RT, followed by a 5 min wash in NS buffer, a 30 sec rinse in 1X NimbleGen final rinse buffer (NimbleGen), and a blow-dry step using high-pressure grade-5 Argon (Badger Welding, Madison, WI). Arrays were scanned using an Axon 4000B scanner at 5 um resolution . Prior to data extraction, images were rotated and doubled in size (without interpolation) using ImageJ software (http://rsb.info.nih.gov/ij/). Features were extracted using GenePix 3.0 software (Axon Instruments, Inc.; Union City, CA), using a fixed feature size. The local background correction from the GenePix software was not applied to raw signal intensities. The values presented are average raw signal intensities.

Disclaimer of Liability

These data are being made available for research purposes only. NimbleGen Systems Inc. does not warrant or assume any legal liability or responsibility for the accuracy, completeness, or usefulness of this data for any purpose.

source_name: mouse spleen mRNA


title: mex67-5TS_v_Mex67WT 061401 slide 3 scan 3a


title: 22-oct-2002-liverE-Mu11KsubBsc

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.9% fish oil (providing 0.5 dietary wt% DHA and 1.0en%). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.
After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hepatic gene expression analysis. From these 5 mice, livers were dissected according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), which consisted of carefully dissecting out the liver, rinsing with 70% EtOH to remove adhering blood, trimming of adhering connective tissue with scissors, cutting into 100-150 mg subsections, freezing in liquid N2, and storing at -80°C until gene expression and lipid analysis. Hepatic lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Liver tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hepatic RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.


title: 22-oct-2002-liverD-Mu11KsubBsc

source_name: liver/hapatocytes


title: S803


title: mex67-5TS_v_Mex67WT 052801 slide 1 scan 3


title: L799

description:
Mouse liver mRNA was isolated from adult Swiss Webster liver tissue obtained from Pel-Freez Biologicals (Rogers, AK). mRNA isolation was performed using oligo-dT hybrid capture on magnetic streptavidin beads using a commercially available kit (PolyATract System 1000, Promega, Madison, WI).  poly A+ RNA was converted to double-stranded cDNA using GibCo (Rockville, MD) BRL’s SuperScript Choice System and an oligo dT primer containing the T7 RNA polymerase promoter (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).  in vitro transcription (IVT) was used to produce biotin-labeled cRNA from the cDNA using the Ambion (Austin, TX) MEGAscript T7 kit. cRNA was purified using Qiagen RNeasy RNA purification columns  according to the manufacturer’s instructions. Before hybridization, cRNA was fragmented to an average size of 50-200 bp. Microarrays were hybridized with 12 ug cRNA in 300 ul, in the presence of 50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20 for 16 hours at 45 degrees C. After hybridization, arrays were washed in non-stringent (NS) buffer (6X SSPE, 0.01% (v/v) Tween 20) for 5 min at RT, followed by washing in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) for 30 min at 45 degrees C.  After washing, arrays were stained with streptavidin-Cy3 conjugate from Amersham Pharmacia for 25 min at RT, followed by a 5 min wash in NS buffer, a 30 sec rinse in 1X NimbleGen final rinse buffer (NimbleGen), and a blow-dry step using high-pressure grade-5 Argon (Badger Welding, Madison, WI). Arrays were scanned using an Axon 4000B scanner at 5 um resolution . Prior to data extraction, images were rotated and doubled in size (without interpolation) using ImageJ software (http://rsb.info.nih.gov/ij/). Features were extracted using GenePix 3.0 software (Axon Instruments, Inc.; Union City, CA), using a fixed feature size. The local background correction from the GenePix software was not applied to raw signal intensities. The values presented are average raw signal intensities.

Disclaimer of Liability

These data are being made available for research purposes only. NimbleGen Systems Inc. does not warrant or assume any legal liability or responsibility for the accuracy, completeness, or usefulness of this data for any purpose.

source_name: mouse liver mRNA


title: WT (BY4741) #1

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: Testis-other tissues

status: Public on Oct 25 2002

description: The testicular RNA was obtained from only one 15-year-old male cynomolgus monkey, and the other RNA was a mixture of RNA obtained from 10 tissues (brain, heart, skin, liver, spleen, renal, pancreas, stomach, small intestine, and large intestine) of two cynomolgus monkeys, a 1-year-old female and a 21-year-old female.  RNA was isolated with Trizol (Life Technologies) and purified with Oligo-Tex (TakaraBio).  Both 0.7 µg mRNA probes were labeled with Cy3- and Cy5- dioxynucleotide (Pharmacia) and co-hybridized to DNA spots. The amount of RNA from each tissue was 0.07 µg in the mixed RNA probe.  After the hybridization and washing procedure, slides were scanned with ScanArray (GSI Inc.).

organism: Macaca fascicularis

source_name: mixture of 10 tissues of cynomolgus monkey (brain, heart, skin, liver, spleen, renal, pancreas, stomach, small intestine, and large intestine)


title: 22-oct-2002-liverA2-Mu11KsubBsc

source_name: liver /hepatocytes


title: 269811CE_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, 15 µg of RNA was used.
Keywords = somatosensory cortex 5-HT2A receptor DOI

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

source_name: Mouse somatosensory cortex 1 hour after saline treatment


title: L796


title: 269801AC_4


title: hsotd_4

status: Public on Feb 25 2002

city: Basel

first: Thomas

last: Loop

address: Klingelbergstrasse 50

department: Institute of Zoology

institute: University of Basel

description:
For overexpression of otd, the hsp-otd line 5A generated by Royet and Finkelstein was used [Royet et al.: Pattern formation in Drosophila head development: the role of the orthodenticle homeobox gene. Development 1995; 121:3561-3572.]. All fly stocks were kept on standard cornmeal/yeast/agar medium at 25°C.  Embryos were collected overnight for 12 hours on grape juice plates, further kept for 4 hours at 25°C and then subjected to a 37°C heat shock for 25 min, followed by a recovery period of 25 min at 25°C before RNA isolation.  Therefore, at the time of RNA isolation these embryos were at embryonic stages 10-17 [Leemans et al.: Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays. Proc Natl Acad Sci U S A 2000; 97:12138-12143.]. Embryos younger than embryonic stage 10 were not used, since heat shock in these earlier stages results in lethality [Walter set al.: Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos. Dev Genet 1990; 11:270-279.]

Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast prep homogenizer FP120 (BIO 101). After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech). cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.
This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was purified by PAGE. For first-strand cDNA synthesis, a typical 40 ul reaction contained 25 ug RNA, 200 pmol T7-(T)24 primer, 500 uM of each dNTP and 800 units reverse transcriptase (AMV Superscript II). The reaction was incubated for 1 h at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 h in a total volume of 340 ul, using 20 units Escherichia coli DNA ligase, 80 units E. coli DNA polymerase I and 4 units RNase H in the presence of 250 uM of each dNTP. After second-strand cDNA synthesis, 0.5 ul RNase A (100 mg/ml) (Qiagen) was added and the samples were incubated at 37°C for 30 min. Thereafter, 7.5 ul proteinase K (10 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min. After cDNA synthesis was completed, samples were phenol-chloroform extracted, using Phase Lock Gel (5 Prime-3 Prime) and ethanol precipitated. Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit: Ambion.). A 20 ul reaction volume contained between 0.3-1.5 ug cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2 ul 10x T7 enzyme mix. The reaction was incubated at 37°C for 8 h. Thereafter, the unincorporated NTPs were removed by running the sample over an RNeasy spin column (Qiagen). Samples were precipitated, taken up in 20 ul DEPC-treated water and spectrophotometrically quantified. Thereafter, 40 ug of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 35 min in a volume of 25 ul, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate. After the fragmentation, the samples were placed on ice.
Gene Chips were pre-hybridized with 220 ul hybridization buffer (1x MES (pH 6.7), 1 M NaCl, 0.01% Triton, 0.5 g/l acetylated BSA, 0.5 g/l sonicated herring sperm DNA) for 15 min at 45°C on a rotisserie (Heidolph) at 60 rpm. Hybridization was done in a final volume of 220 ul hybridization buffer, containing 40 g fragmented biotinylated cRNA. The samples were heated to 95°C for 5 min and briefly spun down. Hybridizations were carried out for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After hybridization, the arrays were briefly rinsed with 6x SSPE-T (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01% Triton) and washed on a Fluidics station (Affymetrix). Hybridized arrays were stained with 220 ul detection solution (1x MES buffer, containing 2.5 ul streptavidin-R phycoerythrin conjugate (1 mg/ml) (Molecular Probes)) and 2.0 mg/ml acetylated BSA (Sigma) at 40°C for 15 min and washed again before being scanned.

The data was normalized against the mean of the total sums of Avg Diff values across all 16 arrays used in this study.

organism: Drosophila melanogaster

source_name: Drosophila melanogaster embryos


title: WT (BY4741) +GlcN (SS) #1

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: Renal cell carcinoma: rccc-26, tumor 2 (part 1 of 2)

status: Public on Jul 19 2001

city: Heidelberg

first: Wolfgang

last: Huber

address: Im Neuenheimer Feld 280

department: Molecular Genome Analysis

country: Germany

institute: German Cancer Research Center

description:
clinical.stage=III
grade.of.malignancy=2
type=clearcell
tumor.site=kidney
multiplefoci=FALSE
survival.months=NA
died=NA
tumorsize.cm=5
comment=The part 2 filters are from batch ReImage3_jan2000. On these, clone 1 has not been spotted in fields 5 and 6. The part 1 filters are from ReImage3_nov99.
sex=m
age=62
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624247 reuse=3 date=14-Jun-2000

source_name: renal clear cell carcinoma (primary tumor)


title: hsotd_1

source_name: Drosophila embryos


title: Renal cell carcinoma: rccc-25, normal 2 (part 1 of 2)

description:
sex=f
age=60
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624244 reuse=5 date=04-Jul-2000

source_name: normal kidney tissue (as reference)


title: Renal cell carcinoma: rccc-24, normal 1 (part 1 of 2)

description:
sex=m
age=36
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3625205 reuse=3 date=04-May-2000


title: Renal cell carcinoma: rccc-26, tumor 1 (part 1 of 2)

description:
clinical.stage=III
grade.of.malignancy=2
type=clearcell
tumor.site=kidney
multiplefoci=FALSE
survival.months=NA
died=NA
tumorsize.cm=5
comment=The part 2 filters are from batch ReImage3_jan2000. On these, clone 1 has not been spotted in fields 5 and 6. The part 1 filters are from ReImage3_nov99.
sex=m
age=62
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624259 reuse=2 date=28-Mar-2000


title: Renal cell carcinoma: rccc-26, normal 1 (part 1 of 2)

description:
sex=m
age=62
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624247 reuse=2 date=28-Mar-2000


title: Renal cell carcinoma: rccc-27, normal 1 (part 1 of 2)

description:
sex=f
age=67
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3625226 reuse=2 date=17-Mar-2000


title: L792


title: Renal cell carcinoma: rccc-23, tumor 2 (part 1 of 2)

description:
clinical.stage=I
grade.of.malignancy=1
type=clearcell
tumor.site=kidney
multiplefoci=FALSE
survival.months=NA
died=NA
tumorsize.cm=7
sex=f
age=47
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624250 reuse=5 date=05-Jul-2000


title: Renal cell carcinoma: rccc-24, tumor 1 (part 1 of 2)

description:
clinical.stage=III
grade.of.malignancy=3
type=clearcell
tumor.site=kidney
multiplefoci=NA
survival.months=NA
died=NA
tumorsize.cm=NA
sex=m
age=36
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3625204 reuse=3 date=04-May-2000


title: Renal cell carcinoma: rccc-26, normal 2 (part 1 of 2)

description:
sex=m
age=62
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624259 reuse=3 date=14-Jun-2000


title: Renal cell carcinoma: rccc-27, tumor 1 (part 1 of 2)

description:
clinical.stage=III
grade.of.malignancy=2
type=clearcell
tumor.site=kidney
multiplefoci=FALSE
survival.months=NA
died=NA
tumorsize.cm=8
sex=f
age=67
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3625227 reuse=2 date=17-Mar-2000


title: Renal cell carcinoma: rccc-25, tumor 2 (part 1 of 2)

description:
clinical.stage=III
grade.of.malignancy=1
type=clearcell
tumor.site=kidney
multiplefoci=FALSE
survival.months=32
died=FALSE
tumorsize.cm=2.7
sex=f
age=60
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624243 reuse=5 date=04-Jul-2000


title: Renal cell carcinoma: rccc-25, normal 1 (part 1 of 2)

description:
sex=f
age=60
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624243 reuse=4 date=19-Apr-2000


title: Renal cell carcinoma: rccc-23, normal 2 (part 1 of 2)

description:
sex=f
age=47
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=2581251 reuse=5 date=05-Jul-2000


title: Renal cell carcinoma: rccc-25, tumor 1 (part 1 of 2)

description:
clinical.stage=III
grade.of.malignancy=1
type=clearcell
tumor.site=kidney
multiplefoci=FALSE
survival.months=32
died=FALSE
tumorsize.cm=2.7
sex=f
age=60
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624244 reuse=4 date=19-Apr-2000


title: Renal cell carcinoma: rccc-23, normal 1 (part 1 of 2)

description:
sex=f
age=47
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624250 reuse=4 date=27-Apr-2000


title: Renal cell carcinoma: rccc-23, tumor 1 (part 1 of 2)

description:
clinical.stage=I
grade.of.malignancy=1
type=clearcell
tumor.site=kidney
multiplefoci=FALSE
survival.months=NA
died=NA
tumorsize.cm=7
sex=f
age=47
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=2581251 reuse=4 date=27-Apr-2000


title: Renal cell carcinoma: rccc-22, tumor 2 (part 1 of 2)

description:
clinical.stage=I
grade.of.malignancy=1
type=clearcell
tumor.site=kidney
multiplefoci=TRUE
survival.months=NA
died=NA
tumorsize.cm=4.5
sex=m
age=44
RNA=poly-A+
For spot quality assessment / error bars, use duplicate spots (each clone is spotted twice) and foreground/background ratio
Keywords = renal clear cell carcinoma
Keywords = kidney
Lot batch = batch=ReImage3_nov99 filter=3624253 reuse=5 date=27-Jun-2000


title: S800

description:
Total mouse spleen RNA was obtained from Ambion, Inc. (Austin, TX). mRNA isolation was performed using oligo-dT hybrid capture on magnetic streptavidin beads using a commercially available kit (PolyATract System 1000, Promega, Madison, WI).  poly A+ RNA was converted to double-stranded cDNA using GibCo (Rockville, MD) BRL’s SuperScript Choice System and an oligo dT primer containing the T7 RNA polymerase promoter (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’).  in vitro transcription (IVT) was used to produce biotin-labeled cRNA from the cDNA using the Ambion (Austin, TX) MEGAscript T7 kit. cRNA was purified using Qiagen RNeasy RNA purification columns  according to the manufacturer’s instructions. Before hybridization, cRNA was fragmented to an average size of 50-200 bp. Microarrays were hybridized with 12 ug cRNA in 300 ul, in the presence of 50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20 for 16 hours at 45 degrees C. After hybridization, arrays were washed in non-stringent (NS) buffer (6X SSPE, 0.01% (v/v) Tween 20) for 5 min at RT, followed by washing in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) for 30 min at 45 degrees C.  After washing, arrays were stained with streptavidin-Cy3 conjugate from Amersham Pharmacia for 25 min at RT, followed by a 5 min wash in NS buffer, a 30 sec rinse in 1X NimbleGen final rinse buffer (NimbleGen), and a blow-dry step using high-pressure grade-5 Argon (Badger Welding, Madison, WI). Arrays were scanned using an Axon 4000B scanner at 5 um resolution . Prior to data extraction, images were rotated and doubled in size (without interpolation) using ImageJ software (http://rsb.info.nih.gov/ij/). Features were extracted using GenePix 3.0 software (Axon Instruments, Inc.; Union City, CA), using a fixed feature size. The local background correction from the GenePix software was not applied to raw signal intensities. The values presented are average raw signal intensities.

Disclaimer of Liability

These data are being made available for research purposes only. NimbleGen Systems Inc. does not warrant or assume any legal liability or responsibility for the accuracy, completeness, or usefulness of this data for any purpose.


title: AJ_HF_s86_Week9

status: Public on Jul 31 2004

first: Jatin

last: Misra

institute: MIT

description:
AJ mouse, total liver RNA, high-fat diet, Week9
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, high-fat diet, Week9

source_name: control, Cy3 dye, mixture of total RNA from liver, skeletal muscle, brain, heart, lung, testes, hypothalamus, brown adipose tissue, white adipose tissue, and kidney of C57 mice'


title: Old Control Day 6 #74 C

status: Public on Oct 05 2003

city: Columbia

first: Scott

last: Pattison

state: MO

institute: University of Missouri

description:
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: Old Day 6 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 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)

source_name: soleus muscle