title: S.l. animal 46, interbout arousal, reverse, brain

description:
Squirrel tissues were rapidly dissected and frozen on liquid nitrogen.
For each tissue sample total RNA was extracted using Trizol (Invitrogen). Fluorescently labelled cDNA was synthesized using amino-allyl adducts coupled to Cy3 and Cy5 fluors (www.microarrays.org) and compared to a reference RNA by hybridization to two arrays with reversal of the labeled fluorophores.  The reference RNA pool was prepared for each tissue by pooling RNA samples isolated from animals sampled under all conditions.  Microarrays were hybridized overnight at 65Ž°C, washed, scanned, and the images scanned and quantified (GenePix 4000A, Axon Instruments, CA).
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 3000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. Without background correction, the log base 10 expression ratios of the median pixel intensities were established, and the spatial and intensity based trends in the data were removed by Lowess normalization (GeneSpring, Silicon Genetics).
Data for each pair of fluor-reversed slides were combined by averaging the ratios derived from the two measurements.  The expression of each cDNA was then normalized to the median ratio observed for that transcript in the summer animals.

source_name: Spermophilus lateralis brain reference RNA

source_name: Animal 46, interbout arousal, reverse, brain


title: S.l. animal 16, late torpor, forward, heart

status: Public on Dec 03 2004

type: RNA

city: Pacific Grove

first: Andrew

last: Gracey

state: CA

country: USA

institute: Stanford University

description:
Squirrel tissues were rapidly dissected and frozen on liquid nitrogen.
For each tissue sample total RNA was extracted using Trizol (Invitrogen). Fluorescently labelled cDNA was synthesized using amino-allyl adducts coupled to Cy3 and Cy5 fluors (www.microarrays.org) and compared to a reference RNA by hybridization to two arrays with reversal of the labeled fluorophores.  The reference RNA pool was prepared for each tissue by pooling RNA samples isolated from animals sampled under all conditions.  Microarrays were hybridized overnight at 65ŽÝC, washed, scanned, and the images scanned and quantified (GenePix 4000A, Axon Instruments, CA).
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 3000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. Without background correction, the log base 10 expression ratios of the median pixel intensities were established, and the spatial and intensity based trends in the data were removed by Lowess normalization (GeneSpring, Silicon Genetics).
Data for each pair of fluor-reversed slides were combined by averaging the ratios derived from the two measurements.  The expression of each cDNA was then normalized to the median ratio observed for that transcript in the summer animals.

molecule: total RNA

organism: Callospermophilus lateralis

source_name: Animal 16, late torpor, forward, heart

source_name: Spermophilus lateralis heart reference RNA


title: S.l. animal 2, summer awake, reverse, heart

source_name: Animal 2, summer awake, reverse, heart


title: S.l. animal 5, summer awake, reverse, brain

source_name: Sample 5, summer awake, reverse, brain


title: S.l. animal 130, interbout arousal, forward, heart

source_name: Animal 130, interbout arousal, forward, heart


title: S.l. animal 41, interbout arousal, reverse, brain

source_name: Animal 41, interbout arousal, reverse, brain


title: S.l. animal 5, summer awake, forward, brain

source_name: Sample 5, summer awake, forward, brain


title: S.l. animal 46, interbout arousal, forward, brain

source_name: Animal 46, interbout arousal, forward, brain


title: S.l. animal 16, late torpor, reverse, heart

source_name: Animal 16, late torpor, reverse, heart


title: S.l. animal 4, summer awake, reverse, brain

source_name: Animal 4, summer awake, reverse, brain


title: S.l. animal 41, interbout arousal, forward, brain

source_name: Animal 41, interbout arousal, forward, brain


title: S.l. animal 5, summer awake, forward, heart

source_name: Animal 5, summer awake, forward, heart


title: S.l. animal 26, late torpor, forward, brain

source_name: Animal 26, late torpor, forward, brain


title: S.l. animal 37, late torpor, forward, brain

source_name: Animal 37, late torpor, forward, brain


title: S.l. animal 2, summer awake, reverse, brain

source_name: Animal 2, summer awake, reverse, brain


title: S.l. animal 16, late torpor, reverse, brain

source_name: Spermophilus lateralis brain reference RNA

source_name: Animal 16, late torpor, reverse, brain


title: S.l. animal 12, late torpor, reverse, brain

source_name: Animal 12, late torpor, reverse, brain


title: S.l. animal 4, summer awake, forward, brain

source_name: Animal 4, summer awake, forward, brain


title: S.l. animal 3, summer awake, forward, brain

source_name: Animal 3, summer awake, forward, brain


title: S.l. animal 2, summer awake, forward, brain

source_name: Animal 2, summer awake, forward, brain


title: S.t. animal 6, summer awake, forward, liver

description:
Squirrel liver tissue was rapidly dissected and frozen on liquid nitrogen.
For each tissue sample total RNA was extracted using Trizol (Invitrogen). Fluorescently labelled cDNA was synthesized using amino-allyl adducts coupled to Cy3 and Cy5 fluors (www.microarrays.org) and compared to a reference RNA by hybridization to two arrays with reversal of the labeled fluorophores.  The reference RNA pool was prepared for each tissue by pooling RNA samples isolated from animals sampled under all conditions.  Microarrays were hybridized overnight at 65ŽÝC, washed, scanned, and the images scanned and quantified (GenePix 4000A, Axon Instruments, CA).
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 3000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. Without background correction, the log base 10 expression ratios of the median pixel intensities were established, and the spatial and intensity based trends in the data were removed by Lowess normalization (GeneSpring, Silicon Genetics).
Data for each pair of fluor-reversed slides were combined by averaging the ratios derived from the two measurements.  The expression of each cDNA was then normalized to the median ratio observed for that transcript in the summer animals.

organism: Ictidomys tridecemlineatus

source_name: Animal 6, summer awake, forward, liver

source_name: Spermophilus lateralis liver reference RNA


title: S.l. animal 3, summer awake, reverse, brain

source_name: Animal 3, summer awake, reverse, brain


title: S.l. animal, 19 late torpor, reverse, brain

source_name: Animal, 19 late torpor, reverse, brain


title: S.l. animal 26, late torpor, reverse, brain

source_name: Animal 26, late torpor, reverse, brain


title: S.t. animal 6, summer awake, reverse, liver

source_name: Animal 6, summer awake, reverse, liver


title: S.l. animal 19, late torpor, forward, brain

source_name: Animal 19, late torpor, forward, brain


title: S.l. animal 16, late torpor, forward, brain

source_name: Animal 16, late torpor, forward, brain


title: S.l. animal 1 summer awake, reverse, brain

source_name: Animal 1 summer awake, reverse, brain


title: S.l. animal 12, late torpor, forward, brain

source_name: Animal 12, late torpor, forward, brain


title: S.t. animal 8, late torpor, forward, liver

source_name: Animal 8, late torpor, forward, liver


title: S.t. animal 5, summer awake, forward, liver

source_name: Animal 5, summer awake, forward, liver


title: S.t. animal 2143, summer awake, reverse, liver

source_name: Animal 2143, summer awake, reverse, liver


title: S.t. animal 8, late torpor, reverse, liver

source_name: Animal 8, late torpor, reverse, liver


title: S.t. animal 2143, summer awake, forward, liver

source_name: Animal 2143, summer awake, forward, liver


title: S.t. animal 5, summer awake, reverse, liver

source_name: Animal 5, summer awake, reverse, liver


title: S.l. animal 1, summer awake, forward, brain

source_name: Animal 1, summer awake, forward, brain

source_name: Spermophilus lateralis brain reference RNA


title: S.t. animal 2142, summer awake, forward, liver

source_name: Animal 2142, summer awake, forward, liver

source_name: Spermophilus lateralis liver reference RNA


title: S.t. animal 1930, late torpor, forward, liver

source_name: Animal 1930, late torpor, forward, liver


title: S.t. animal 1930, late torpor, reverse, liver

source_name: Animal 1930, late torpor, reverse, liver


title: S.t. animal 2142, summer awake, reverse, liver

source_name: Animal 2142, summer awake, reverse, liver


title: S.t. animal 1922, interbout arousal, forward, liver

source_name: Animal 1922, interbout arousal, forward, liver


title: S.t. animal 1904, late torpor, forward, liver

source_name: Animal 1904, late torpor, forward, liver


title: S.t. animal 1906, interbout arousal, forward, liver

source_name: Animal 1906, interbout arousal, forward, liver


title: S.t. animal 1906, interbout arousal, reverse, liver

source_name: Animal 1906, interbout arousal, reverse, liver


title: S.l. animal 3, summer awake, forward, liver

description:
Squirrel liver tissue was rapidly dissected and frozen on liquid nitrogen.
For each tissue sample total RNA was extracted from tissues using Trizol (Invitrogen). Fluorescently labelled cDNA was synthesized using amino-allyl adducts coupled to Cy3 and Cy5 fluors (www.microarrays.org) and compared to a reference RNA by hybridization to two arrays with reversal of the labeled fluorophores.  The reference RNA pool was prepared for each tissue by pooling RNA samples isolated from animals sampled under all conditions.  Microarrays were hybridized overnight at 65Ž°C, washed, scanned, and the images scanned and quantified (GenePix 4000A, Axon Instruments, CA).
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 3000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. Without background correction, the log base 10 expression ratios of the median pixel intensities were established, and the spatial and intensity based trends in the data were removed by Lowess normalization (GeneSpring, Silicon Genetics).
Data for each pair of fluor-reversed slides were combined by averaging the ratios derived from the two measurements.  The expression of each cDNA was then normalized to the median ratio observed for that transcript in the summer animals.

source_name: Animal 3, summer awake, forward, liver


title: S.t. animal 1912, interbout arousal, forward, liver

source_name: Animal 1912, interbout arousal, forward, liver


title: S.t. animal 1912, interbout arousal, reverse, liver

source_name: Animal 1912, interbout arousal, reverse, liver


title: S.t. animal 1831, late torpor, reverse, liver

source_name: Animal 1831, late torpor, reverse, liver


title: S.t. animal 1922, interbout arousal, reverse, liver

source_name: Animal 1922, interbout arousal, reverse, liver


title: S.l. animal 37, late torpor, reverse, brain

source_name: Animal 37, late torpor, reverse, brain


title: S.t. animal 1902, late torpor, reverse, liver

source_name: Animal 1902, late torpor, reverse, liver


title: S.t. animal 1831, late torpor, forward, liver

source_name: Animal 1831, late torpor, forward, liver


title: S.t. animal 1904, late torpor, reverse, liver

source_name: Animal 1904, late torpor, reverse, liver


title: S.t. animal 1902, late torpor, forward, liver

source_name: Animal 1902, late torpor, forward, liver

source_name: Spermophilus lateralis liver reference RNA


title: 150657

status: Public on Dec 01 2004

city: Iowa City

first: Rui

last: Zhao

state: IA

department: Biological Sciences

institute: The University of Iowa

description:
opaque a/a cells of strain P37005 and opaque alpha/alpha cells of strain WO-1, individually grown to stationary phase in modified Lee's medium at 25ºC for 48 hr
cells pelleted and resuspended in fresh modified Lee's medium at original stationary phase cell concentration under following conditions
1) homogeneous a/a cells, 3.5 hr
2) homogeneous alpha/alpha cells, 3.5 hr
3) mixed a/a-alpha/alpha cell cultures, 3.5 hr and 7.5 hr
The cDNA from target-source1 and target-source2 was indirectly coupled separately with Cy5 (1) and Cy3 (2) monoreactive fluors, respectively, in 0.05 M sodium bicarbonate, pH 9.0. The Cy5 and Cy3 labelled cDNAs were purified with a QIAquick PCR purification kit, quantified, combined, and suspended in 30 µl of hybridization buffer (10X SSC and 0.2% SDS) and applied to microarray slides
Fluorescent array images were collected for both Cy5 and Cy3 channels with a GenePix 4000B fluorescent scanner, image intensity data was extracted with Genepix Pro 4.0

organism: Candida albicans

source_name: homogeneous alpha/alpha cells, 3.5 hr

source_name: homogeneous a/a cells, 3.5 hr


title: 150655

source_name: mixed a/a-alpha/alpha cell cultures, 3.5 hr


title: 150653


title: 150654


title: S.t. animal 10, late torpor, forward, liver

source_name: Animal 10, late torpor, forward, liver


title: 150646


title: 150652


title: 150656


title: 150647


title: 150636

source_name: mixed a/a-alpha/alpha cell cultures, 7.5 hr


title: 150645


title: 150630


title: 150610


title: 150618


title: 150626


title: 150627


title: 150637


title: 150611


title: 150609


title: S.t. animal 10, late torpor, reverse, liver

source_name: Animal 10, late torpor, reverse, liver


title: 150607


title: 150644


title: 150606


title: 150603

source_name: mixed a/a-alpha/alpha cell culture, 7.5 hr


title: 150604


title: 150619


title: BoneMarrow_77A01

status: Public on Nov 22 2004

city: Los Angeles

first: Christopher

last: Lee

department: Dept. of Chemistry & Biochemistry

institute: University of California, Los Angeles

description:
Bone marrow total RNA was ordered from Clontech (Palo Alto, CA). Total RNA was extracted from 200-400 mg of frozen tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was additionally purified with the RNAeasy kit (Qiagen, Valencia, CA). Labeled cDNA for array hybridizations was generated by linear amplification of total RNA, followed by direct labeling of the amplification products. Briefly, RNA samples from five normal tissues were individually amplified using the BD SMARTTM mRNA amplification kit (BD Biosciences, Palo Alto, CA). 1.5 ug of total RNA from each normal tissue was used as a substrate for amplification according to manufacturer’s specifications. Each amplified sample was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to check the quality and integrity of the mRNA. Typically, the amplification products migrated as a broad peak, with the majority of transcripts ranging from 500 to 4000 bp. The amplified mRNAs were prepared for hybridization as follows: a pool of all the samples to be hybridized to the splicing arrays was assembled by mixing equal amounts of mRNA from each of the five tissue samples and four glioblastoma (GBM) samples (not discussed in this study). This comparator pool was used for all hybridizations. For each hybridization, 250 ng of the amplified normal tissue mRNA or GBM mRNA and 250 ng of the pool were labeled with Cyanine 5-dCTP and Cyanine 3-dCTP, respectively (Perkin Elmer, Boston, MA) using the Agilent Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA). Dye-swapped labeling reactions were performed in parallel and hybridized to the second array on the slide. Labeling reactions were carried out according to the manufacturer’s specifications, with the following modification: instead of the DNA primer provided with the kit, 2 ug of random hexamers (MWG Biotech, High Point, NC) were used to prime the reaction. The entire Cyanine 5 and Cyanine 3 labeling reactions were combined according to the Fluorescent Direct Label kit protocol and were prepared for hybridization to the array using the In situ Hybridization kit Plus (Agilent Technologies, Palo Alto, CA) according to manufacturer’s specifications, with the exception that hybridizations were performed for 36-48 hrs. Slides were washed and dried according to manufacturer’s specifications and scanned on an Agilent Microarray Scanner (Model G2565BA; Agilent Technologies, Palo Alto, CA). Scanned images were analyzed using the Agilent Feature Extraction Software (Version A.7.1.1; Agilent Technologies, Palo Alto, CA).
Keywords = Alternative Splicing
Keywords = Agilent

organism: Homo sapiens

source_name: Bone Marrow

source_name: Pool (mixing equal amounts of brain, muscle, liver, bone marrow, testes and four glioblastoma (GBM) samples)


title: BoneMarrow_66A01


title: 150602


title: 150631


title: Muscle_73A01

description:
Muscle tissue samples were obtained from the University of Maryland Brain and Tissue Bank. Total RNA was extracted from 200-400 mg of frozen tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was additionally purified with the RNAeasy kit (Qiagen, Valencia, CA). Labeled cDNA for array hybridizations was generated by linear amplification of total RNA, followed by direct labeling of the amplification products. Briefly, RNA samples from five normal tissues were individually amplified using the BD SMARTTM mRNA amplification kit (BD Biosciences, Palo Alto, CA). 1.5 ug of total RNA from each normal tissue was used as a substrate for amplification according to manufacturer’s specifications. Each amplified sample was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to check the quality and integrity of the mRNA. Typically, the amplification products migrated as a broad peak, with the majority of transcripts ranging from 500 to 4000 bp. The amplified mRNAs were prepared for hybridization as follows: a pool of all the samples to be hybridized to the splicing arrays was assembled by mixing equal amounts of mRNA from each of the five tissue samples and four glioblastoma (GBM) samples (not discussed in this study). This comparator pool was used for all hybridizations. For each hybridization, 250 ng of the amplified normal tissue mRNA or GBM mRNA and 250 ng of the pool were labeled with Cyanine 5-dCTP and Cyanine 3-dCTP, respectively (Perkin Elmer, Boston, MA) using the Agilent Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA). Dye-swapped labeling reactions were performed in parallel and hybridized to the second array on the slide. Labeling reactions were carried out according to the manufacturer’s specifications, with the following modification: instead of the DNA primer provided with the kit, 2 ug of random hexamers (MWG Biotech, High Point, NC) were used to prime the reaction. The entire Cyanine 5 and Cyanine 3 labeling reactions were combined according to the Fluorescent Direct Label kit protocol and were prepared for hybridization to the array using the In situ Hybridization kit Plus (Agilent Technologies, Palo Alto, CA) according to manufacturer’s specifications, with the exception that hybridizations were performed for 36-48 hrs. Slides were washed and dried according to manufacturer’s specifications and scanned on an Agilent Microarray Scanner (Model G2565BA; Agilent Technologies, Palo Alto, CA). Scanned images were analyzed using the Agilent Feature Extraction Software (Version A.7.1.1; Agilent Technologies, Palo Alto, CA).
Keywords = Alternative Splicing
Keywords = Agilent

source_name: Muscle


title: BoneMarrow_77A02


title: Muscle_62A01


title: Muscle_62A02


title: 150600


title: Ambrosino 2

city: Glasgow

first: Tomoko

last: Iwata

address: Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden

department: Division of Cancer Sciences & Molecular Pathology

country: United Kingdom

institute: Faculty of Medicine, University of Glasgow

description:
Hybridizations were performed in triplicate, using total of 7 RNA samples extracted from 3 independent batches of proliferating cardiomyocyte cell line derived from p38alpha knockout mouse.

Total RNA was extracted with RNA Clean (Hybaid), according to the manufacturer’s instruction and further purified with RNeasy mini kit (Qiagen). Direct labeling of 20 µg of total RNA primed with Oligo(dT) was performed in the presence of Cy-5 and Cy-3-dUTP (Amersham Pharmacia Biotech) using SuperScript II (Invitrogen). The Cy5- and Cy3-labelled cDNA samples were then mixed and purified with GFX DNA purification kit (Amersham Pharmacia Biotech). Prior to hybridization, samples were supplemented with 1.2 µg of salmon sperm DNA and 5 µg of Poly(dA) and dried in SpeedVac at 60°C. Array slides were pre-hybridized in 6x SSC, 0.5% SDS, 1% BSA at 42°C for 1 h and incubated in a boiled water for 2 min just prior to hybridization. The cDNA samples were resuspended in 15 µl of hybridization buffer (50% formamide, 6x SSC, 0.5% SDS and 5%Denhardt’s) and denatured by incubating at 95°C for 2 min. Hybridization was at 42oC for 16 h The slides were washed for 10 min each with 2 x SSC, then with 0.5 x SSC, 0.1% SDS, followed by 0.1 x SSC, 0.1% SDS at room temperature. Scanning was performed with GenePix 4000B (Axon). The data acquisition and initial data analysis was performed with GenePix Pro 3.0 and the data tables were analyzed further in Excel in order to obtain the gene list. Quality control was performed to confirm scanner alignment and absence of significant bubbles and scratches. Scatter plots were further used to eliminate the unacceptable hybridization data. The multiple spike-in control RNA templates were added to the sample upon direct labeling, and the successful labeling and hybridization was confirmed in each hybridizations. Twelve housekeeping genes in the array (GAPDH, HPRT and S16, S9, and S8 ribosomal proteins) gave average value of Ratio of Medians (ROM), 1.30 ± 0.26, which was confirmed to be acceptable. GenePix Pro program calculates the Normalization Factor, based on the premise that the arithmetic mean of the ratios from every feature on the given array should be equal to 1. Normalization was therefore performed by multiplying the Factor to ROM in each gene. The program also identifies features that did not give good alignment to the expected spotted area as “flagged” spots, indicative of the impaired-quality hybridization of the specific genes. All flagged genes were removed from the list before further analysis.

Keywords = NIA mouse 15K
Keywords = cardiomyocytes
Keywords = p38 MAPK

organism: Mus musculus

source_name: immortalized cardiomyocyte culture derived from p38alpha MAPK knockout mouse

source_name: immortalized cardiomyocyte culture derived from wild type control mouse


title: Muscle_73A02

description:
Muscle tissue samples were obtained from the University of Maryland Brain and Tissue Bank. Total RNA was extracted from 200-400 mg of frozen tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was additionally purified with the RNAeasy kit (Qiagen, Valencia, CA). Labeled cDNA for array hybridizations was generated by linear amplification of total RNA, followed by direct labeling of the amplification products. Briefly, RNA samples from five normal tissues were individually amplified using the BD SMARTTM mRNA amplification kit (BD Biosciences, Palo Alto, CA). 1.5 ?g of total RNA from each normal tissue was used as a substrate for amplification according to manufacturer’s specifications. Each amplified sample was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to check the quality and integrity of the mRNA. Typically, the amplification products migrated as a broad peak, with the majority of transcripts ranging from 500 to 4000 bp. The amplified mRNAs were prepared for hybridization as follows: a pool of all the samples to be hybridized to the splicing arrays was assembled by mixing equal amounts of mRNA from each of the five tissue samples and four glioblastoma (GBM) samples (not discussed in this study). This comparator pool was used for all hybridizations. For each hybridization, 250 ng of the amplified normal tissue mRNA or GBM mRNA and 250 ng of the pool were labeled with Cyanine 5-dCTP and Cyanine 3-dCTP, respectively (Perkin Elmer, Boston, MA) using the Agilent Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA). Dye-swapped labeling reactions were performed in parallel and hybridized to the second array on the slide. Labeling reactions were carried out according to the manufacturer’s specifications, with the following modification: instead of the DNA primer provided with the kit, 2 ?g of random hexamers (MWG Biotech, High Point, NC) were used to prime the reaction. The entire Cyanine 5 and Cyanine 3 labeling reactions were combined according to the Fluorescent Direct Label kit protocol and were prepared for hybridization to the array using the In situ Hybridization kit Plus (Agilent Technologies, Palo Alto, CA) according to manufacturer’s specifications, with the exception that hybridizations were performed for 36-48 hrs. Slides were washed and dried according to manufacturer’s specifications and scanned on an Agilent Microarray Scanner (Model G2565BA; Agilent Technologies, Palo Alto, CA). Scanned images were analyzed using the Agilent Feature Extraction Software (Version A.7.1.1; Agilent Technologies, Palo Alto, CA).
Keywords = Alternative Splicing
Keywords = Agilent


title: BoneMarrow_66A02


title: 150608


title: Liver_76A02

description:
Liver tissue samples were obtained from the UCLA Tissue Bank. Total RNA was extracted from 200-400 mg of frozen tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was additionally purified with the RNAeasy kit (Qiagen, Valencia, CA). Labeled cDNA for array hybridizations was generated by linear amplification of total RNA, followed by direct labeling of the amplification products. Briefly, RNA samples from five normal tissues were individually amplified using the BD SMARTTM mRNA amplification kit (BD Biosciences, Palo Alto, CA). 1.5 ?g of total RNA from each normal tissue was used as a substrate for amplification according to manufacturer’s specifications. Each amplified sample was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to check the quality and integrity of the mRNA. Typically, the amplification products migrated as a broad peak, with the majority of transcripts ranging from 500 to 4000 bp. The amplified mRNAs were prepared for hybridization as follows: a pool of all the samples to be hybridized to the splicing arrays was assembled by mixing equal amounts of mRNA from each of the five tissue samples and four glioblastoma (GBM) samples (not discussed in this study). This comparator pool was used for all hybridizations. For each hybridization, 250 ng of the amplified normal tissue mRNA or GBM mRNA and 250 ng of the pool were labeled with Cyanine 5-dCTP and Cyanine 3-dCTP, respectively (Perkin Elmer, Boston, MA) using the Agilent Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA). Dye-swapped labeling reactions were performed in parallel and hybridized to the second array on the slide. Labeling reactions were carried out according to the manufacturer’s specifications, with the following modification: instead of the DNA primer provided with the kit, 2 ?g of random hexamers (MWG Biotech, High Point, NC) were used to prime the reaction. The entire Cyanine 5 and Cyanine 3 labeling reactions were combined according to the Fluorescent Direct Label kit protocol and were prepared for hybridization to the array using the In situ Hybridization kit Plus (Agilent Technologies, Palo Alto, CA) according to manufacturer’s specifications, with the exception that hybridizations were performed for 36-48 hrs. Slides were washed and dried according to manufacturer’s specifications and scanned on an Agilent Microarray Scanner (Model G2565BA; Agilent Technologies, Palo Alto, CA). Scanned images were analyzed using the Agilent Feature Extraction Software (Version A.7.1.1; Agilent Technologies, Palo Alto, CA).
Keywords = Alternative Splicing
Keywords = Agilent

source_name: Liver


title: Ambrosino 1


title: Brain_75A02

description:
Brain tissue samples were obtained from the University of Maryland Brain and Tissue Bank.  Total RNA was extracted from 200-400 mg of frozen tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was additionally purified with the RNAeasy kit (Qiagen, Valencia, CA). Labeled cDNA for array hybridizations was generated by linear amplification of total RNA, followed by direct labeling of the amplification products. Briefly, RNA samples from five normal tissues were individually amplified using the BD SMARTTM mRNA amplification kit (BD Biosciences, Palo Alto, CA). 1.5 ?g of total RNA from each normal tissue was used as a substrate for amplification according to manufacturer’s specifications. Each amplified sample was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to check the quality and integrity of the mRNA. Typically, the amplification products migrated as a broad peak, with the majority of transcripts ranging from 500 to 4000 bp. The amplified mRNAs were prepared for hybridization as follows: a pool of all the samples to be hybridized to the splicing arrays was assembled by mixing equal amounts of mRNA from each of the five tissue samples and four glioblastoma (GBM) samples (not discussed in this study). This comparator pool was used for all hybridizations. For each hybridization, 250 ng of the amplified normal tissue mRNA or GBM mRNA and 250 ng of the pool were labeled with Cyanine 5-dCTP and Cyanine 3-dCTP, respectively (Perkin Elmer, Boston, MA) using the Agilent Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA). Dye-swapped labeling reactions were performed in parallel and hybridized to the second array on the slide. Labeling reactions were carried out according to the manufacturer’s specifications, with the following modification: instead of the DNA primer provided with the kit, 2 ?g of random hexamers (MWG Biotech, High Point, NC) were used to prime the reaction. The entire Cyanine 5 and Cyanine 3 labeling reactions were combined according to the Fluorescent Direct Label kit protocol and were prepared for hybridization to the array using the In situ Hybridization kit Plus (Agilent Technologies, Palo Alto, CA) according to manufacturer’s specifications, with the exception that hybridizations were performed for 36-48 hrs. Slides were washed and dried according to manufacturer’s specifications and scanned on an Agilent Microarray Scanner (Model G2565BA; Agilent Technologies, Palo Alto, CA). Scanned images were analyzed using the Agilent Feature Extraction Software (Version A.7.1.1; Agilent Technologies, Palo Alto, CA).
Keywords = Alternative Splicing
Keywords = Agilent

source_name: Brain


title: Liver_65A01


title: Liver_76A01


title: Ambrosino 3

description:
Hybridizations were performed in triplicate, using total of 7 RNA samples extracted from 3 independent batches of proliferating cardiomyocyte cell line derived from p38alpha knockout mouse.

Total RNA was extracted with RNA Clean (Hybaid), according to the manufacturer’s instruction and further purified with RNeasy mini kit (Qiagen). Direct labeling of 20 µg of total RNA primed with Oligo(dT) was performed in the presence of Cy-5 and Cy-3-dUTP (Amersham Pharmacia Biotech) using SuperScript II (Invitrogen). The Cy5- and Cy3-labelled cDNA samples were then mixed and purified with GFX DNA purification kit (Amersham Pharmacia Biotech). Prior to hybridization, samples were supplemented with 1.2 µg of salmon sperm DNA and 5 µg of Poly(dA) and dried in SpeedVac at 60°C. Array slides were pre-hybridized in 6x SSC, 0.5% SDS, 1% BSA at 42°C for 1 h and incubated in a boiled water for 2 min just prior to hybridization. The cDNA samples were resuspended in 15 µl of hybridization buffer (50% formamide, 6x SSC, 0.5% SDS and 5%Denhardt’s) and denatured by incubating at 95°C for 2 min. Hybridization was at 42oC for 16 h The slides were washed for 10 min each with 2 x SSC, then with 0.5 x SSC, 0.1% SDS, followed by 0.1 x SSC, 0.1% SDS at room temperature. Scanning was performed with GenePix 4000B (Axon). The data acquisition and initial data analysis was performed with GenePix Pro 3.0 and the data tables were analyzed further in Excel in order to obtain the gene list. Quality control was performed to confirm scanner alignment and absence of significant bubbles and scratches. Scatter plots were further used to eliminate the unacceptable hybridization data. The multiple spike-in control RNA templates were added to the sample upon direct labeling, and the successful labeling and hybridization was confirmed in each hybridizations. Twelve housekeeping genes in the array (GAPDH, HPRT and S16, S9, and S8 ribosomal proteins) gave average value of Ratio of Medians (ROM), 1.30 ± 0.26, which was confirmed to be acceptable. GenePix Pro program calculates the Normalization Factor, based on the premise that the arithmetic mean of the ratios from every feature on the given array should be equal to 1. Normalization was therefore performed by multiplying the Factor to ROM in each gene. The program also identifies features that did not give good alignment to the expected spotted area as “flagged” spots, indicative of the impaired-quality hybridization of the specific genes. All flagged genes were removed from the list before further analysis.

Keywords = NIA mouse 15K
Keywords = cardiomyocytes
Keywords = p38alpha MAPK


title: Brain_64A01


title: 150605


title: Testes_74A02

description:
Testes tissue samples were obtained from the UCLA Tissue Bank. Total RNA was extracted from 200-400 mg of frozen tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was additionally purified with the RNAeasy kit (Qiagen, Valencia, CA). Labeled cDNA for array hybridizations was generated by linear amplification of total RNA, followed by direct labeling of the amplification products. Briefly, RNA samples from five normal tissues were individually amplified using the BD SMARTTM mRNA amplification kit (BD Biosciences, Palo Alto, CA). 1.5 ?g of total RNA from each normal tissue was used as a substrate for amplification according to manufacturer’s specifications. Each amplified sample was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to check the quality and integrity of the mRNA. Typically, the amplification products migrated as a broad peak, with the majority of transcripts ranging from 500 to 4000 bp. The amplified mRNAs were prepared for hybridization as follows: a pool of all the samples to be hybridized to the splicing arrays was assembled by mixing equal amounts of mRNA from each of the five tissue samples and four glioblastoma (GBM) samples (not discussed in this study). This comparator pool was used for all hybridizations. For each hybridization, 250 ng of the amplified normal tissue mRNA or GBM mRNA and 250 ng of the pool were labeled with Cyanine 5-dCTP and Cyanine 3-dCTP, respectively (Perkin Elmer, Boston, MA) using the Agilent Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA). Dye-swapped labeling reactions were performed in parallel and hybridized to the second array on the slide. Labeling reactions were carried out according to the manufacturer’s specifications, with the following modification: instead of the DNA primer provided with the kit, 2 ?g of random hexamers (MWG Biotech, High Point, NC) were used to prime the reaction. The entire Cyanine 5 and Cyanine 3 labeling reactions were combined according to the Fluorescent Direct Label kit protocol and were prepared for hybridization to the array using the In situ Hybridization kit Plus (Agilent Technologies, Palo Alto, CA) according to manufacturer’s specifications, with the exception that hybridizations were performed for 36-48 hrs. Slides were washed and dried according to manufacturer’s specifications and scanned on an Agilent Microarray Scanner (Model G2565BA; Agilent Technologies, Palo Alto, CA). Scanned images were analyzed using the Agilent Feature Extraction Software (Version A.7.1.1; Agilent Technologies, Palo Alto, CA).
Keywords = Alternative Splicing
Keywords = Agilent

source_name: Testes


title: Testes_73A01


title: Testes_74A01

description: Testes tissue samples were obtained from the UCLA Tissue Bank. Total RNA was extracted from 200-400 mg of frozen tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.  Total RNA was additionally purified with the RNAeasy kit (Qiagen, Valencia, CA).  Labeled cDNA for array hybridizations was generated by linear amplification of total RNA, followed by direct labeling of the amplification products. Briefly, RNA samples from five normal tissues were individually amplified using the BD SMARTTM mRNA amplification kit (BD Biosciences, Palo Alto, CA).  1.5 ug of total RNA from each normal tissue was used as a substrate for amplification according to manufacturer’s specifications.  Each amplified sample was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to check the quality and integrity of the mRNA.  Typically, the amplification products migrated as a broad peak, with the majority of transcripts ranging from 500 to 4000 bp.  The amplified mRNAs were prepared for hybridization as follows: a pool of all the samples to be hybridized to the splicing arrays was assembled by mixing equal amounts of mRNA from each of the five tissue samples and four glioblastoma (GBM) samples (not discussed in this study).  This comparator pool was used for all hybridizations.  For each hybridization, 250 ng of the amplified normal tissue mRNA or GBM mRNA and 250 ng of the pool were labeled with Cyanine 5-dCTP and Cyanine 3-dCTP, respectively (Perkin Elmer, Boston, MA) using the Agilent Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA).  Dye-swapped labeling reactions were performed in parallel and hybridized to the second array on the slide.  Labeling reactions were carried out according to the manufacturer’s specifications, with the following modification: instead of the DNA primer provided with the kit, 2 ug of random hexamers (MWG Biotech, High Point, NC) were used to prime the reaction.  The entire Cyanine 5 and Cyanine 3 labeling reactions were combined according to the Fluorescent Direct Label kit protocol and were prepared for hybridization to the array using the In situ Hybridization kit Plus (Agilent Technologies, Palo Alto, CA) according to manufacturer’s specifications, with the exception that hybridizations were performed for 36-48 hrs.  Slides were washed and dried according to manufacturer’s specifications and scanned on an Agilent Microarray Scanner (Model G2565BA; Agilent Technologies, Palo Alto, CA). Scanned images were analyzed using the Agilent Feature Extraction Software (Version A.7.1.1; Agilent Technologies, Palo Alto, CA).


title: Brain_64A02


title: Testes_63A02


title: Normal Rat Retinal Müller Cells (biological replicate 3)

status: Public on Nov 17 2004

city: Boston

first: Chiara

last: Gerhardinger

state: MA

address: 20 Staniford Street

institute: Schepens Eye Research Institute

description: Rat Retinal Müller cells from healthy rats.  Müller cells were isolated from the pooled retinas of 12 rats.

organism: Rattus norvegicus

source_name: Sprague-Dawley Rat (male)


title: Diabetic Rat Retinal Müller Cells (technical replicate 2)

description: Rat Retinal Müller cells from diabetic rats (diabetes duration 6 months).  Diabetes was induced by streptozotozine.  Diabetic rats were treated with small doses of insulin to prevent catabolism.  Müller cells were isolated from the pooled retinas of 12 rats.


title: Diabetic Rat Retinal Müller Cells


title: 150601


title: Normal Rat Retinal Müller Cells (biological replicate 2)


title: P7_2

status: Public on Nov 15 2004

city: La Jolla

first: Steven

last: Head

address: 10550 N Torrey Pines Rd

institute: The Scripps Research Institute

description:
Mouse strain = BALB/c
Age: Postnatal day 7 (P7)
Tissue was extracted and pooled from a single litter of 5-7 pups and used for each replicate P7 sample
Total RNA was extracted using RNeasy Midi Kit, according to manufacturerÆs instructions (Qiagen).  The quality of the samples was checked with an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Standard Affymetrix Protocol was used for RNA sample labeling with ENZO BioArray HighYield RNA Transcript Labeling Kit
Keywords = Cerebellum, microarray, development, glycoconjugates, glycosyltransferases, proteoglycans, gene expression
Lot batch =

source_name: Cerebellum


title: Normal Rat Retinal Müller Cells


title: P7_1


title: A_3

description:
Mouse strain = BALB/c
Age: Adult (>30 postnatal)
One adult animal (postnatal 30 day or beyond) was used for each replicate adult sample
Total RNA was extracted using RNeasy Midi Kit, according to manufacturerÆs instructions (Qiagen).  The quality of the samples was checked with an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Standard Affymetrix Protocol was used for RNA sample labeling with ENZO BioArray HighYield RNA Transcript Labeling Kit
Keywords = Cerebellum, microarray, development, glycoconjugates, glycosyltransferases, proteoglycans, gene expression
Lot batch =


title: Brain_75A01


title: A_1


title: YSX3_xylose_oxygen_limited

status: Public on Nov 13 2004

city: Madison

first: Thomas

middle: William

last: Jeffries

state: WI

address: One Gifford Pinchot Drive

institute: Institute for Microbial and Biochemical Technology

description:
S. cerevisiae YSX3 (MATalpha, leu2::LEU2-XYL1, ura3::URA3-XYL2, Ty3::NEO-XYL3) was used in this study. Cells were grown under aerobic or oxygen-limited conditions with glucose or xylose (four conditions). Cells were cultivated aerobically in 200 ml of YP medium with either 20 g/L of glucose or xylose in 1000 ml flasks shaken at 300 rpm and harvested at OD600 = 1. Oxygen-limited cells were cultivated in 50 ml of YP medium with either 40 g/L of glucose or xylose in 125 ml flasks shaken at 100 rpm and harvested at OD600 = 30. For monitoring transcripts in the respiration-deficient mutant, FPL-YSX3P, cells were first grown on YP medium with glucose and harvested at OD600 = 1. The harvested cells were transferred into YP medium with xylose and then RNA was extracted after 24 hours incubation. GeneChip arrays (Affymetrix, Santa Clara, CA) were used to monitor mRNA transcripts of putative S. cerevisiae open reading frames (ORFs). Quantitative RT-PCR was used to measure expression of P. stipitis XYL1, XYL2, and XYL3 transcripts. Total RNA from yeast cells was isolated as described in Holstege et al. cDNA was synthesized with a T7-(dT)24 primer (GENSET Corp). Labeling of RNA transcripts, hybridization, and scanning were performed according to the manufacturer’s instructions (Affymetrix).
Keywords = xylose
Keywords = glucose
Keywords = aerobic
Keywords = oxygen limited

organism: Saccharomyces cerevisiae

source_name: Total RNA


title: YSX3_glucose_oxygen_limited


title: Normal vs. Subtracted 2

status: Public on Nov 16 2004

city: St. Paul

first: Cheryl

last: Dvorak

state: MN

address: 1971 Commonwealth Avenue

department: Veterinary and Biomedical Sciences

institute: University of Minnesota

description:
Porcine jejunal intestinal tissue was obtained from 5-9 week old Yorkshire pigs as described previously (Green et al., 2003). Jejunal segments containing a Peyer's patch were stripped of the underlying smooth muscle layer and stored in RNAlater (Ambion, Inc., Austin, TX) immediately or mounted in an Ussing flux chamber (Green et al., 2003). Treatments were added to the luminal side of the Ussing chamber in both the presence and absence of 50 µM cycloheximide and incubated for 3 hours. Stimulation conditions consisted of (1) log phase Salmonella choleraesuis vaccine strain SC-54 (Roof and Doitchinoff, 1995) at an initial bacterial concentration of 2.7 x 104 CFU/ml; (2) 1 µg/ml lipopolysaccharide and 50 ng/ml cholera toxin (CT+LPS); (3) 100 nM phorbol myristate acetate, 1 mM 8-bromo-cyclic AMP and 5 µg/ml concanavalin A (PBC); and (4) incubated with no stimulation (normal). After the 3 hour incubation, tissues were removed, the region of tissue containing the Peyer's patch was excised and was stored in RNAlater (Ambion, Inc., Austin, TX) at Ð20¡C.
RNA was isolated from all Peyer's patches using Qiagen RNeasy kits (Qiagen Inc., Valencia, CA) and examined for quality using the RNA 6000 Nano LabChip Kit on an Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. Libraries were created in the pCMV¥Sport 6 plasmid using the SuperScript Plasmid System with Gateway Technology (Invitrogen Corporation, Carlsbad, CA) with a modified Not I d(T) primer (5' GACTAGTTCTAGATCGCGAGCGGCCGCCT17VN 3'). The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA.
cRNA was created from each of the subtracted, normal, and immune stimulated libraries following the MAXIscript in vitro transcription kit protocol (Ambion Inc., Austin, TX). The above cRNA's (200 ng) or isolated total RNA (10 ug) were used as templates for the reverse transcription and amino allyl coupling reaction. Either the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) was used for the reverse transcription reaction and clean-up steps or a similar homemade procedure was performed. The microarray chips were incubated at 63¡C for 5 to 16 hours, washed, dried, and scanned using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Images were quantified with QuantArray software version 3 (PerkinElmer, Boston, MA) using the adaptive method to calculate the mean spot intensity and mean background intensity.
Sample_dye_swap: Normal (Cy5)/ Subtracted (Cy3)
Keywords = jejunal Peyer's patch
Keywords = pig
Keywords = small intestine
Lot batch =

organism: Sus scrofa

source_name: subtracted library cRNA

source_name: normal library cRNA


title: YSX3_xylose_petite


title: Sub vs. Sub, 200 ng

description:
Porcine jejunal intestinal tissue was obtained from 5-9 week old Yorkshire pigs as described previously (Green et al., 2003). Jejunal segments containing a Peyer's patch were stripped of the underlying smooth muscle layer and stored in RNAlater (Ambion, Inc., Austin, TX) immediately or mounted in an Ussing flux chamber (Green et al., 2003). Treatments were added to the luminal side of the Ussing chamber in both the presence and absence of 50 µM cycloheximide and incubated for 3 hours. Stimulation conditions consisted of (1) log phase Salmonella choleraesuis vaccine strain SC-54 (Roof and Doitchinoff, 1995) at an initial bacterial concentration of 2.7 x 104 CFU/ml; (2) 1 µg/ml lipopolysaccharide and 50 ng/ml cholera toxin (CT+LPS); (3) 100 nM phorbol myristate acetate, 1 mM 8-bromo-cyclic AMP and 5 µg/ml concanavalin A (PBC); and (4) incubated with no stimulation (normal). After the 3 hour incubation, tissues were removed, the region of tissue containing the Peyer's patch was excised and was stored in RNAlater (Ambion, Inc., Austin, TX) at Ð20¡C.
RNA was isolated from all Peyer's patches using Qiagen RNeasy kits (Qiagen Inc., Valencia, CA) and examined for quality using the RNA 6000 Nano LabChip Kit on an Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. Libraries were created in the pCMV¥Sport 6 plasmid using the SuperScript Plasmid System with Gateway Technology (Invitrogen Corporation, Carlsbad, CA) with a modified Not I d(T) primer (5' GACTAGTTCTAGATCGCGAGCGGCCGCCT17VN 3'). The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA.
cRNA was created from each of the subtracted, normal, and immune stimulated libraries following the MAXIscript in vitro transcription kit protocol (Ambion Inc., Austin, TX). The above cRNA's (200 ng) or isolated total RNA (10 ug) were used as templates for the reverse transcription and amino allyl coupling reaction. Either the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) was used for the reverse transcription reaction and clean-up steps or a similar homemade procedure was performed. The microarray chips were incubated at 63¡C for 5 to 16 hours, washed, dried, and scanned using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Images were quantified with QuantArray software version 3 (PerkinElmer, Boston, MA) using the adaptive method to calculate the mean spot intensity and mean background intensity.
Keywords = jejunal Peyer's patch
Keywords = pig
Keywords = small intestine
Lot batch =


title: Immune Stim. vs. Subtracted

description:
Porcine jejunal intestinal tissue was obtained from 5-9 week old Yorkshire pigs as described previously (Green et al., 2003). Jejunal segments containing a Peyer's patch were stripped of the underlying smooth muscle layer and stored in RNAlater (Ambion, Inc., Austin, TX) immediately or mounted in an Ussing flux chamber (Green et al., 2003). Treatments were added to the luminal side of the Ussing chamber in both the presence and absence of 50 µM cycloheximide and incubated for 3 hours. Stimulation conditions consisted of (1) log phase Salmonella choleraesuis vaccine strain SC-54 (Roof and Doitchinoff, 1995) at an initial bacterial concentration of 2.7 x 104 CFU/ml; (2) 1 µg/ml lipopolysaccharide and 50 ng/ml cholera toxin (CT+LPS); (3) 100 nM phorbol myristate acetate, 1 mM 8-bromo-cyclic AMP and 5 µg/ml concanavalin A (PBC); and (4) incubated with no stimulation (normal). After the 3 hour incubation, tissues were removed, the region of tissue containing the Peyer's patch was excised and was stored in RNAlater (Ambion, Inc., Austin, TX) at Ð20¡C.
RNA was isolated from all Peyer's patches using Qiagen RNeasy kits (Qiagen Inc., Valencia, CA) and examined for quality using the RNA 6000 Nano LabChip Kit on an Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. Libraries were created in the pCMV¥Sport 6 plasmid using the SuperScript Plasmid System with Gateway Technology (Invitrogen Corporation, Carlsbad, CA) with a modified Not I d(T) primer (5' GACTAGTTCTAGATCGCGAGCGGCCGCCT17VN 3'). The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA.
cRNA was created from each of the subtracted, normal, and immune stimulated libraries following the MAXIscript in vitro transcription kit protocol (Ambion Inc., Austin, TX). The above cRNA's (200 ng) or isolated total RNA (10 ug) were used as templates for the reverse transcription and amino allyl coupling reaction. Either the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) was used for the reverse transcription reaction and clean-up steps or a similar homemade procedure was performed. The microarray chips were incubated at 63¡C for 5 to 16 hours, washed, dried, and scanned using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Images were quantified with QuantArray software version 3 (PerkinElmer, Boston, MA) using the adaptive method to calculate the mean spot intensity and mean background intensity.
Sample_dye_swap: Immune Stim. (Cy3)/ Subtracted (Cy5)
Keywords = jejunal Peyer's patch
Keywords = pig
Keywords = small intestine
Lot batch =

source_name: Immune Stimulated library cRNA


title: Sub vs. Sub, 2ug


title: Normal vs. Subtracted

description:
Porcine jejunal intestinal tissue was obtained from 5-9 week old Yorkshire pigs as described previously (Green et al., 2003). Jejunal segments containing a Peyer's patch were stripped of the underlying smooth muscle layer and stored in RNAlater (Ambion, Inc., Austin, TX) immediately or mounted in an Ussing flux chamber (Green et al., 2003). Treatments were added to the luminal side of the Ussing chamber in both the presence and absence of 50 µM cycloheximide and incubated for 3 hours. Stimulation conditions consisted of (1) log phase Salmonella choleraesuis vaccine strain SC-54 (Roof and Doitchinoff, 1995) at an initial bacterial concentration of 2.7 x 104 CFU/ml; (2) 1 µg/ml lipopolysaccharide and 50 ng/ml cholera toxin (CT+LPS); (3) 100 nM phorbol myristate acetate, 1 mM 8-bromo-cyclic AMP and 5 µg/ml concanavalin A (PBC); and (4) incubated with no stimulation (normal). After the 3 hour incubation, tissues were removed, the region of tissue containing the Peyer's patch was excised and was stored in RNAlater (Ambion, Inc., Austin, TX) at Ð20¡C.
RNA was isolated from all Peyer's patches using Qiagen RNeasy kits (Qiagen Inc., Valencia, CA) and examined for quality using the RNA 6000 Nano LabChip Kit on an Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. Libraries were created in the pCMV¥Sport 6 plasmid using the SuperScript Plasmid System with Gateway Technology (Invitrogen Corporation, Carlsbad, CA) with a modified Not I d(T) primer (5' GACTAGTTCTAGATCGCGAGCGGCCGCCT17VN 3'). The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA.
cRNA was created from each of the subtracted, normal, and immune stimulated libraries following the MAXIscript in vitro transcription kit protocol (Ambion Inc., Austin, TX). The above cRNA's (200 ng) or isolated total RNA (10 ug) were used as templates for the reverse transcription and amino allyl coupling reaction. Either the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) was used for the reverse transcription reaction and clean-up steps or a similar homemade procedure was performed. The microarray chips were incubated at 63¡C for 5 to 16 hours, washed, dried, and scanned using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Images were quantified with QuantArray software version 3 (PerkinElmer, Boston, MA) using the adaptive method to calculate the mean spot intensity and mean background intensity.
Sample_dye_swap: Normal (Cy3)/subtracted (Cy5)
Keywords = jejunal Peyer's patch
Keywords = pig
Keywords = small intestine
Lot batch =

source_name: Normal library cRNA


title: A_2


title: P7_3


title: Immune Stim. vs. Subtracted 2

description:
Porcine jejunal intestinal tissue was obtained from 5-9 week old Yorkshire pigs as described previously (Green et al., 2003). Jejunal segments containing a Peyer's patch were stripped of the underlying smooth muscle layer and stored in RNAlater (Ambion, Inc., Austin, TX) immediately or mounted in an Ussing flux chamber (Green et al., 2003). Treatments were added to the luminal side of the Ussing chamber in both the presence and absence of 50 µM cycloheximide and incubated for 3 hours. Stimulation conditions consisted of (1) log phase Salmonella choleraesuis vaccine strain SC-54 (Roof and Doitchinoff, 1995) at an initial bacterial concentration of 2.7 x 104 CFU/ml; (2) 1 µg/ml lipopolysaccharide and 50 ng/ml cholera toxin (CT+LPS); (3) 100 nM phorbol myristate acetate, 1 mM 8-bromo-cyclic AMP and 5 µg/ml concanavalin A (PBC); and (4) incubated with no stimulation (normal). After the 3 hour incubation, tissues were removed, the region of tissue containing the Peyer's patch was excised and was stored in RNAlater (Ambion, Inc., Austin, TX) at Ð20¡C.
RNA was isolated from all Peyer's patches using Qiagen RNeasy kits (Qiagen Inc., Valencia, CA) and examined for quality using the RNA 6000 Nano LabChip Kit on an Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. Libraries were created in the pCMV¥Sport 6 plasmid using the SuperScript Plasmid System with Gateway Technology (Invitrogen Corporation, Carlsbad, CA) with a modified Not I d(T) primer (5' GACTAGTTCTAGATCGCGAGCGGCCGCCT17VN 3'). The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA.
cRNA was created from each of the subtracted, normal, and immune stimulated libraries following the MAXIscript in vitro transcription kit protocol (Ambion Inc., Austin, TX). The above cRNA's (200 ng) or isolated total RNA (10 ug) were used as templates for the reverse transcription and amino allyl coupling reaction. Either the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) was used for the reverse transcription reaction and clean-up steps or a similar homemade procedure was performed. The microarray chips were incubated at 63¡C for 5 to 16 hours, washed, dried, and scanned using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Images were quantified with QuantArray software version 3 (PerkinElmer, Boston, MA) using the adaptive method to calculate the mean spot intensity and mean background intensity.
Sample_dye_swap: Immune Stim. (Cy5)/ Subtracted (Cy3)
Keywords = jejunal Peyer's patch
Keywords = pig
Keywords = small intestine
Lot batch =

source_name: Subtracted library cRNA


title: Immune Stim. vs. Normal

description:
Porcine jejunal intestinal tissue was obtained from 5-9 week old Yorkshire pigs as described previously (Green et al., 2003). Jejunal segments containing a Peyer's patch were stripped of the underlying smooth muscle layer and stored in RNAlater (Ambion, Inc., Austin, TX) immediately or mounted in an Ussing flux chamber (Green et al., 2003). Treatments were added to the luminal side of the Ussing chamber in both the presence and absence of 50 µM cycloheximide and incubated for 3 hours. Stimulation conditions consisted of (1) log phase Salmonella choleraesuis vaccine strain SC-54 (Roof and Doitchinoff, 1995) at an initial bacterial concentration of 2.7 x 104 CFU/ml; (2) 1 µg/ml lipopolysaccharide and 50 ng/ml cholera toxin (CT+LPS); (3) 100 nM phorbol myristate acetate, 1 mM 8-bromo-cyclic AMP and 5 µg/ml concanavalin A (PBC); and (4) incubated with no stimulation (normal). After the 3 hour incubation, tissues were removed, the region of tissue containing the Peyer's patch was excised and was stored in RNAlater (Ambion, Inc., Austin, TX) at Ð20¡C.
RNA was isolated from all Peyer's patches using Qiagen RNeasy kits (Qiagen Inc., Valencia, CA) and examined for quality using the RNA 6000 Nano LabChip Kit on an Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. Libraries were created in the pCMV¥Sport 6 plasmid using the SuperScript Plasmid System with Gateway Technology (Invitrogen Corporation, Carlsbad, CA) with a modified Not I d(T) primer (5' GACTAGTTCTAGATCGCGAGCGGCCGCCT17VN 3'). The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA.
cRNA was created from each of the subtracted, normal, and immune stimulated libraries following the MAXIscript in vitro transcription kit protocol (Ambion Inc., Austin, TX). The above cRNA's (200 ng) or isolated total RNA (10 ug) were used as templates for the reverse transcription and amino allyl coupling reaction. Either the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) was used for the reverse transcription reaction and clean-up steps or a similar homemade procedure was performed. The microarray chips were incubated at 63¡C for 5 to 16 hours, washed, dried, and scanned using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Images were quantified with QuantArray software version 3 (PerkinElmer, Boston, MA) using the adaptive method to calculate the mean spot intensity and mean background intensity.
Sample_dye_swap: Immune Stim. (Cy3)/ Normal (Cy5)
Keywords = jejunal Peyer's patch
Keywords = pig
Keywords = small intestine
Lot batch =


title: Immune Stim. vs. Normal2

description:
Porcine jejunal intestinal tissue was obtained from 5-9 week old Yorkshire pigs as described previously (Green et al., 2003). Jejunal segments containing a Peyer's patch were stripped of the underlying smooth muscle layer and stored in RNAlater (Ambion, Inc., Austin, TX) immediately or mounted in an Ussing flux chamber (Green et al., 2003). Treatments were added to the luminal side of the Ussing chamber in both the presence and absence of 50 µM cycloheximide and incubated for 3 hours. Stimulation conditions consisted of (1) log phase Salmonella choleraesuis vaccine strain SC-54 (Roof and Doitchinoff, 1995) at an initial bacterial concentration of 2.7 x 104 CFU/ml; (2) 1 µg/ml lipopolysaccharide and 50 ng/ml cholera toxin (CT+LPS); (3) 100 nM phorbol myristate acetate, 1 mM 8-bromo-cyclic AMP and 5 µg/ml concanavalin A (PBC); and (4) incubated with no stimulation (normal). After the 3 hour incubation, tissues were removed, the region of tissue containing the Peyer's patch was excised and was stored in RNAlater (Ambion, Inc., Austin, TX) at Ð20¡C.
RNA was isolated from all Peyer's patches using Qiagen RNeasy kits (Qiagen Inc., Valencia, CA) and examined for quality using the RNA 6000 Nano LabChip Kit on an Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. Libraries were created in the pCMV¥Sport 6 plasmid using the SuperScript Plasmid System with Gateway Technology (Invitrogen Corporation, Carlsbad, CA) with a modified Not I d(T) primer (5' GACTAGTTCTAGATCGCGAGCGGCCGCCT17VN 3'). The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA.
cRNA was created from each of the subtracted, normal, and immune stimulated libraries following the MAXIscript in vitro transcription kit protocol (Ambion Inc., Austin, TX). The above cRNA's (200 ng) or isolated total RNA (10 ug) were used as templates for the reverse transcription and amino allyl coupling reaction. Either the SuperScript Indirect cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA) was used for the reverse transcription reaction and clean-up steps or a similar homemade procedure was performed. The microarray chips were incubated at 63¡C for 5 to 16 hours, washed, dried, and scanned using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Images were quantified with QuantArray software version 3 (PerkinElmer, Boston, MA) using the adaptive method to calculate the mean spot intensity and mean background intensity.
Sample_dye_swap: Immune Stim. (Cy5)/ Normal (Cy3)
Keywords = jejunal Peyer's patch
Keywords = pig
Keywords = small intestine
Lot batch =


title: YSX3_glucose_petite


title: YSX3_xylose_aerobic


title: YSX3_glucose_aerobic


title: Sub vs. Sub, 500ng


title: Ifh1+(3)

status: Public on Nov 10 2004

city: Geneva

first: Stephan

middle: B.

last: Schawalder

address: Quai Ansermet 30

department: Departement of Molecular biology

country: Switzerland

institute: University of Geneva

description:
Experiment design
-Type of experiment: genome wide binding analysis
-Experimental factor: genotype (Wild-Type, Fhl1-myc, Ifh1-myc); drug (rapamycin)
-Number of hybridization performed in the experiment: 15 (four conditions with three to five biological replicates each)
-Reference used for each condition: ChIP from the wild-type strain
-Quality control: one dye-swap per condition

Samples used, extract preparation and labeling
-The origin of the biological sample:
o	Species: S. cerevisiae
o	Cell type: (1) W303; (2) W303+IFH1-13myc-TRP1MX6;(3) W303+FHL1-13myc-HIS3MX6.
-Growth conditions: log phase (2x107/mL) at 30ºC in YPD medium; when indicated rapamycin was added at the final concentration of 200 ng/mL.
-Cross-linking for 30 min at 30ºC
-Chromatin immunoprecipitation: Dynabeads M280 coupled to sheep anti-mouse IgG or sheep anti-rabbit IgG and mouse monoclonal antibodies against the myc epitope (culture supernatant of 9E10) were used for immunoprecipitation according to standard techniques.
-Labeling protocol(s): each IP was blunt-ended, ligated to linker DNA, then subjected to Ligation Mediated (LM)-PCR in the presence of amino-allyl dUTP. Labeling was performed for 1 hour at room temperature in the presence of NHS-ester Cy3 or Cy5 in 0.1 M Sodium Carbonate. The labeling reaction was stopped with 2 M Hydroxylamine and the labeled DNA purified.

Hybridization procedures and parameters
-Standard protocols were used

Measurement data and specifications
-Software for scanning: GenePix Pro 4.0 (Axon Instruments, Inc.)
-Scanner: GenePix 4000A (Axon Instruments, Inc.)
Keywords = Genome-wide binding of Fhl1 and Ifh1


title: Ifh1-(2)


title: Ifh1+(1)


title: Ifh1+(2)


title: Ifh1-(1)


title: Fhl1+(4)


title: Fhl1+(5)


title: Ifh1-(4)


title: DBA_subB

status: Public on Nov 09 2004

city: Berlin

first: Soner

last: Aksu

country: Germany

institute: Humboldt-Universität zu Berlin

description:
Pooled RNA samples were prepared by epididymal fat tissues of 42 days old 20 male DBA animals. Pooling was used to reduce individual variability, which is high in outbred populations. For the hybridization of the tissue specific RNAs to the GeneChips, samples were prepared according to the recommendations of the Affymetrix user guide. First strand synthesis was carried out by a T7-(dT)24 primer and SuperScript II Reverse Transcriptase (Gibco BRL, Life Technologies GmbH, Eggenstein, Germany) using 10 mg whole RNA sample. Second strand synthesis was done according to the SuperScript Choice System (Gibco BRL, Life Technologies GmbH) by E. coli DNA-Polymerase I, E. coli Ligase and RNaseH. Fragment end-polishing was performed using T4-Polymerase. An in vitro transcription reaction was used to incorporate Biotin-11-CTP and Biotin-16-UTP to the cRNA probe (BioArray HighYield RNA Transcript Labeling Kit, Enzo). The fragmented cDNA was hybridized overnight at 45°C to ensure the quality of the probe. The expression levels were calculated with the GeneChip Expression Analysis softwares Data Mining Tool (Version 1.2.) and Microarray Suite (version 4.0.1.) provided by Affymetrix. Initially, hybridization signals on every GeneChip were scaled using all probe sets to minimize differences in overall signal intensities between two arrays allowing more reliable detection of biologically relevant changes in the sample. The Affymetrix decision matrix was used for the assessment of present, marginally present, and absent transcript amounts of a gene in a tested RNA.
Keywords = adiposity
Keywords = selected mouse lines
Keywords = diabetes
Keywords = insulin
Keywords = leptin

source_name: epididymal fat tissue


title: Fhl1-(2)


title: DU6i_subB

description:
Pooled RNA samples were prepared by epididymal fat tissues of 42 days old 20 male DU6i animals. Pooling was used to reduce individual variability, which is high in outbred populations. For the hybridization of the tissue specific RNAs to the GeneChips, samples were prepared according to the recommendations of the Affymetrix user guide. First strand synthesis was carried out by a T7-(dT)24 primer and SuperScript II Reverse Transcriptase (Gibco BRL, Life Technologies GmbH, Eggenstein, Germany) using 10 mg whole RNA sample. Second strand synthesis was done according to the SuperScript Choice System (Gibco BRL, Life Technologies GmbH) by E. coli DNA-Polymerase I, E. coli Ligase and RNaseH. Fragment end-polishing was performed using T4-Polymerase. An in vitro transcription reaction was used to incorporate Biotin-11-CTP and Biotin-16-UTP to the cRNA probe (BioArray HighYield RNA Transcript Labeling Kit, Enzo). The fragmented cDNA was hybridized overnight at 45°C to ensure the quality of the probe. The expression levels were calculated with the GeneChip Expression Analysis softwares Data Mining Tool (Version 1.2.) and Microarray Suite (version 4.0.1.) provided by Affymetrix. Initially, hybridization signals on every GeneChip were scaled using all probe sets to minimize differences in overall signal intensities between two arrays allowing more reliable detection of biologically relevant changes in the sample. The Affymetrix decision matrix was used for the assessment of present, marginally present, and absent transcript amounts of a gene in a tested RNA.
Keywords = adiposity
Keywords = selected mouse lines
Keywords = diabetes
Keywords = insulin
Keywords = leptin


title: Fhl1+(2)


title: Ifh1-(3)


title: Fhl1-(3)


title: DU6_subB

description:
Pooled RNA samples were prepared by epididymal fat tissues of 42 days old 20 male DU6 animals. Pooling was used to reduce individual variability, which is high in outbred populations. For the hybridization of the tissue specific RNAs to the GeneChips, samples were prepared according to the recommendations of the Affymetrix user guide. First strand synthesis was carried out by a T7-(dT)24 primer and SuperScript II Reverse Transcriptase (Gibco BRL, Life Technologies GmbH, Eggenstein, Germany) using 10 mg whole RNA sample. Second strand synthesis was done according to the SuperScript Choice System (Gibco BRL, Life Technologies GmbH) by E. coli DNA-Polymerase I, E. coli Ligase and RNaseH. Fragment end-polishing was performed using T4-Polymerase. An in vitro transcription reaction was used to incorporate Biotin-11-CTP and Biotin-16-UTP to the cRNA probe (BioArray HighYield RNA Transcript Labeling Kit, Enzo). The fragmented cDNA was hybridized overnight at 45°C to ensure the quality of the probe. The expression levels were calculated with the GeneChip Expression Analysis softwares Data Mining Tool (Version 1.2.) and Microarray Suite (version 4.0.1.) provided by Affymetrix. Initially, hybridization signals on every GeneChip were scaled using all probe sets to minimize differences in overall signal intensities between two arrays allowing more reliable detection of biologically relevant changes in the sample. The Affymetrix decision matrix was used for the assessment of present, marginally present, and absent transcript amounts of a gene in a tested RNA.
Keywords = adiposity
Keywords = selected mouse lines
Keywords = diabetes
Keywords = insulin
Keywords = leptin


title: Fhl1+(3)


title: Fhl1+(1)


title: DUKs_subB

description:
Pooled RNA samples were prepared by epididymal fat tissues of 42 days old 20 male DUKs animals. Pooling was used to reduce individual variability, which is high in outbred populations. For the hybridization of the tissue specific RNAs to the GeneChips, samples were prepared according to the recommendations of the Affymetrix user guide. First strand synthesis was carried out by a T7-(dT)24 primer and SuperScript II Reverse Transcriptase (Gibco BRL, Life Technologies GmbH, Eggenstein, Germany) using 10 mg whole RNA sample. Second strand synthesis was done according to the SuperScript Choice System (Gibco BRL, Life Technologies GmbH) by E. coli DNA-Polymerase I, E. coli Ligase and RNaseH. Fragment end-polishing was performed using T4-Polymerase. An in vitro transcription reaction was used to incorporate Biotin-11-CTP and Biotin-16-UTP to the cRNA probe (BioArray HighYield RNA Transcript Labeling Kit, Enzo). The fragmented cDNA was hybridized overnight at 45°C to ensure the quality of the probe. The expression levels were calculated with the GeneChip Expression Analysis softwares Data Mining Tool (Version 1.2.) and Microarray Suite (version 4.0.1.) provided by Affymetrix. Initially, hybridization signals on every GeneChip were scaled using all probe sets to minimize differences in overall signal intensities between two arrays allowing more reliable detection of biologically relevant changes in the sample. The Affymetrix decision matrix was used for the assessment of present, marginally present, and absent transcript amounts of a gene in a tested RNA.
Keywords = adiposity
Keywords = selected mouse lines
Keywords = diabetes
Keywords = insulin
Keywords = leptin


title: Fhl1-(1)


title: Dog3 Left Lung (Injured) Base Dependent

city: Baltimore

first: Shwu-Fan

last: Ma

state: MD

address: 5501 Hopkins Bayview Circle

institute: Johns Hopkins University

description:
3 mongrel dogs (weight 21.3±1.5 kg) were anesthetized with 25 mk/kg pentobarbital i.v. and instrumented with femoral arterial and venous catheters. Anesthesia was maintained with additional pentobarbital (5 mg/kg iv every hour and when indicated) and muscle relaxation provided by pancuronium (3 mg bolus and 0.5 mg hourly iv). A 39 or
41 french double-lumen endobronchial tube was placed via a tracheostomy and position confirmed by fiberoptic bronchoscopy. The animals were ventilated with a “dual piston” large animal ventilator, permitting independent control of tidal volume (Vt), inspired oxygen fraction (FiO2), and positive end-expiratory pressure (PEEP), and measurement of airway opening pressure (Paw) and end-tidal partial pressure of carbon dioxide (ETPCO2) for each lung. Oxygen saturation (SaO2) was continuously measured using a pulse oximeter applied to the tongue or ear and ETPCO2, arterial blood pressure (Pa), Paw, and esophageal pressure (Pes) were continuously recorded. An infusion of lactated ringers (LR, 5-10 ml/kg/hr) was given for maintenance fluid replacement. Rectal or PA temperature was maintained at 36±1?C with radiant heat lamps. At the conclusion of the study, the animals were sacrificed by exsanguination after supplemental pentobarbital (10 mg/kg i.v.). After instrumentation, the individual lung Vt !Sample_description =were adjusted to provide an ETPCO2 of 30-35 mmHg at a respiratory rate of 25 breaths/min. The left lung was then mildly injured by repeated lavage with warmed saline, 20 ml/kg repeated 4 times. Ventilation continued at baseline settings, with the addition of 5 cm H2O PEEP to the control right lung, for 5 hours. At that time the animal was sacrificed by exsanguination, the chest opened, and 10 tissue samples taken from 5 corresponding
regions in both lungs (apex- dependent and non-dependent, base- non dependent, mid, and dependent). The lung tissue samples were immersed in RNAlater (Ambion, Austin, Texas) and frozen for subsequent analysis. In some animals, additional tissue samples were taken and stored in 10% formalin for immunohistochemistry.
Sample_hybridization: total RNA was converted to first-strand cDNA using a hybrid reverse transcription primer consisting of oligo-dT and T7 RNA polymerase promoter sequences. The single-stranded cDNA was then converted to double-stranded cDNA. Complementary DNA corresponding to 5-10 µg of total RNA was used in a cRNA amplification step using T7 RNA polymerase and two biotinylated nucleotide precursors. The resulting biotinylated cRNA was fragmented to a size of approximately 50 bp, and approximately 20-30 µg of the biotinylated cRNA was hybridized to the Human HG_U133A GeneChip (Affymetrix, Santa Clara, CA). The bound cRNA was visualized by binding of streptavidin/phycoerythrin conjugates to the hybridized GeneChip, followed by laser scanning of bound phycoerythrin.
Sample_RNA_Isolation: Tissues (~50 mg) were taken frozen and directly solubilized in chaotropic solubilization buffer using a Brinkman Polytron tissue disruptor. Larger tissue fragments (>100mg) were pulverized into frozen powder with a mortar and pestle, pre-chilled to liquid nitrogen temperature, and then the frozen powder was solubilized with the Polytron. RNA was purified using Trizol LS (Life Technologies) and an additional RNA purification step was conducted using the RNAeasy purification kit (Qiagen Inc., Valencia, CA). Approximately 10 µg of purified, total RNA was used for analyses.
Sample_strain: Mongrel
Keywords = unilateral canine lung injury model, microarray, gene expression, mechanical ventilation

organism: Canis lupus familiaris

source_name: dog lungs


title: Dog3 Left Lung (Injured) Base NonDependent


title: Dog3 Right Lung (NonInjured) Base NonDependent


title: Dog3 Right Lung (NonInjured) Base Dependent


title: Dog3 Left Lung (Injured) Apex Dependent


title: Dog3 Left Lung (Injured) Apex NonDependent


title: Dog2 Left Lung (Injured) Base NonDependent


title: Dog2 Left Lung (Injured) Base Dependent


title: Dog3 Right Lung (NonInjured) Apex Dependent


title: DUKs_subA


title: Dog3 Right Lung (NonInjured) Apex NonDependent


title: Dog1 Left Lung (Injured) Base Dependent


title: Dog2 Right Lung (NonInjured) Apex Dependent


title: DBA_subA


title: Dog2 Right Lung (NonInjured) Base NonDependent


title: Dog1 Left Lung (Injured) Base NonDependent


title: Dog2 Right Lung (NonInjured) Base Dependent


title: Dog2 Right Lung (NonInjured) Apex NonDependent


title: Dog2 Left Lung (Injured) Apex NonDependent


title: Dog1 Right Lung (NonInjured) Base Dependent


title: DU6_subA


title: Dog1 Left Lung (Injured) Apex NonDependent


title: DU6i_subA


title: Dog1 Right Lung (NonInjured) Apex NonDependent


title: Dog2 Left Lung (Injured) Apex Dependent


title: Dog1 Left Lung (Injured) Apex Dependent


title: Dog1 Right Lung (NonInjured) Base NonDependent


title: Dog1 Right Lung (NonInjured) Apex Dependent


title: 002A-750610_TestA

status: Public on Nov 03 2004

city: University Park

first: Frank

last: Pugh

state: PA

address: 452 North Frear Laboratory

department: Biochemistry and Molecular Biology

institute: The Pennsylvania State University

description:
Strains were grown in YPD media at 25ûC to OD600 = ~0.8. Then the cultures were shifted to 37ûC for 15 min. ( by adding an equal volume of heated media for the heat shocked set), or mock treated at 25ûC for 15 min. (by adding an equal volume of 25ûC media for the mock treated set).  After 15 min. incubation, cells were harvested at 25ûC.  See methods and publication for details on RNA extraction and microarray hybridization.
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software.
Keywords = Saccharomyces cerevisiae

source_name: Null (BY4741) Heat Shocked cDNA (CH1)

source_name: Null (BY4741) Mock Treated cDNA (CH2)


title: 001A-750700_HomotypicA

source_name: Null (BY4741) Mock Treated cDNA (CH1)


title: 001B-730620_HomotypicB


title: CEA_SGF:ALY041_ASP

status: Public on Nov 01 2004

city: EVRY

first: Michèle

middle: T

last: Martin

address: 2, rue Gaston Crémieux

department: DSV/DRR

country: France

institute: CEA

description:
Human mammary skin biopsies were obtained from plastic surgery, from the Tissue Bank for Research (AFM-BTR, hôpital La Pitié Salpétrière, Paris) and keratinocytes were isolated with trypsin. Primary cultures were initiated in complete KGM2 semi-defined medium (Clonetics) on flasks coated with collagen type I (Falcon Biocoat). Sub-confluent cultures were re-plated to allow amplification through a second passage. Third passage cultures were seeded at 40 000 cells/cm2 and were grown at 37°C and 5% CO2. Cultures were confluent at day 5 and cells were further incubated for 3 days post-confluence in order to reach a morphological differentiated state.
Differentiated cultures were then irradiated with a 60 Co source at two different dose rates: 28 cGy/min and 2 cGy/min to deliver 10 mGy and 2 Gy, respectively. Sham irradiated cells received the same treatment, except for irradiation. Cultures were then returned to the incubator and harvested at 3,6,15,24,48 and 72 hours following irradiation in order to prepare RNA extracts. Media was changed the day before irradiation and renewed every two days. Total RNA was amplified using the MessageAmp RNA Kit (Ambion). One round of amplification was performed. Amplified RNA was labeled by coupling with aminoallyl-modified dUTP (Sigma), followed by a subsequent incorporation of monofunctionnal Cy3 or Cy5-N-succinimide esters (Amersham Biosciences). For each labeling experiment, amplified RNA (2 µg) was reverse-trancribed using random hexamers in the presence of 200 µM aminoallyl-dUTP, 500 µM each dATP, dCTP, dGTP, 300 µM dTTP, 400 U of Superscript II polymerase (Invitrogen) 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 H (Invitrogen) and incubating at 37°C for 15 minutes. The volume was adjusted to 400 µl with H20, aminoallyl-cDNA was purified by centrifugation in a microcon (YM-30 Millipore Amicon), eluted with 20 µl of water and then dried in a vacuum to 10µl. One µl sodium bicarbonate 0.5 M was added to aminoallyl-cDNA and subsequently mixed with vacuum-dried monofunctional dye. Coupling reaction was performed during 1 hour at room temperature in the dark. Labeled cDNAs were purified with a nucleospin column (Macherey Nagel), 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 1 volume of 5 M ammonium acetate in chilled ethanol.
The microarray slides were pre-hybridized in 3,5x SSC, 0,1% SDS and 1% BSA for 30 mn at 50°C. The labeled extract pellet was suspended in the hybridization solution (formamide 50%, 2,5x Denhardt, 0,5% SDS, 0,6% SSPE). The solution was heated to 95°C, added onto the prehybridized microarray slides, and hybridized at 42°C in a humidified chamber for 16 to 18 hours. Microarray glass slides were washed with 0,1X SSC / 0,1 % SDS at room temperature for 15 minutes and then twice with 0,1X SSC in the same conditions.

source_name: Differentiated primary keratinocyte culture, 48 hours, 0 Gy

source_name: Differentiated primary keratinocyte culture, 48 hours, 2 Gy


title: CEA_SGF:ALY040_ASP

source_name: Differentiated primary keratinocyte culture, 48 hours, 10 mGy


title: CEA_SGF:ALY042_ASP


title: CEA_SGF:ALY038_ASP

source_name: Differentiated primary keratinocyte culture, 15 hours, 10 mGy

source_name: Differentiated primary keratinocyte culture, 15 hours, 0 Gy


title: CEA_SGF:ALY037_ASP


title: CEA_SGF:ALY039_ASP


title: CEA_SGF:ALY036_ASP

source_name: Differentiated primary keratinocyte culture, 6 hours, 10 mGy

source_name: Differentiated primary keratinocyte culture, 6 hours, 0 Gy


title: CEA_SGF:ALY029_ASP

source_name: Differentiated primary keratinocyte culture, 72 hours, 0 Gy

source_name: Differentiated primary keratinocyte culture, 72 hours, 2 Gy


title: CEA_SGF:ALY033_ASP

source_name: Differentiated primary keratinocyte culture, 3 hours, 0 Gy

source_name: Differentiated primary keratinocyte culture, 3 hours, 2 Gy


title: CEA_SGF:ALY034_ASP


title: CEA_SGF:ALY031_ASP

source_name: Differentiated primary keratinocyte culture, 3 hours, 10 mGy


title: CEA_SGF:ALY035_ASP


title: CEA_SGF:ALY032_ASP


title: CEA_SGF:ALY024_ASP


title: CEA_SGF:ALY027_ASP

source_name: Differentiated primary keratinocyte culture, 15 hours, 2 Gy


title: CEA_SGF:ALY030_ASP


title: CEA_SGF:ALY022_ASP


title: CEA_SGF:ALY025_ASP


title: CEA_SGF:ALY021_ASP


title: CEA_SGF:ALY023_ASP


title: CEA_SGF:ALY028_ASP

source_name: Differentiated primary keratinocyte culture, 72 hours, 10 mGy


title: CEA_SGF:ALY020_ASP


title: CEA_SGF:ALY026_ASP


title: CEA_SGF:ALY018_ASP


title: 002B-720630_TestB

source_name: Null (BY4741) Heat Shocked cDNA (CH2)


title: CEA_SGF:ALY016_ASP


title: CEA_SGF:ALY017_ASP


title: CEA_SGF:ALY010_ASP


title: CEA_SGF:ALE042_ASP


title: CEA_SGF:ALY009_ASP

source_name: Differentiated primary keratinocyte culture, 24 hours, 10 mGy

source_name: Differentiated primary keratinocyte culture, 24h hours, 0 Gy


title: CEA_SGF:ALY012_ASP

source_name: Differentiated primary keratinocyte culture, 48 hours, 0 Gy


title: CEA_SGF:ALE031_ASP


title: CEA_SGF:ALE037_ASP


title: CEA_SGF:ALY015_ASP


title: CEA_SGF:ALE027_ASP


title: CEA_SGF:ALY014_ASP


title: CEA_SGF:ALY008_ASP


title: CEA_SGF:ALE032_ASP

source_name: Differentiated primary keratinocyte culture, 24 hours, 2 Gy


title: CEA_SGF:ALY013_ASP

source_name: Differentiated primary keratinocyte culture, 48 hours, 2 Gy


title: CEA_SGF:ALE036_ASP


title: CEA_SGF:ALE030_ASP


title: CEA_SGF:ALE034_ASP


title: CEA_SGF:ALE035_ASP


title: CEA_SGF:ALE033_ASP


title: CEA_SGF:ALE026_ASP


title: CEA_SGF:ALE010_ASP


title: CEA_SGF:AMD041_ASP

description:
Human mammary skin biopsies were obtained from plastic surgery, from the Tissue Bank for Research (AFM-BTR, hôpital La Pitié Salpétrière, Paris) and keratinocytes were isolated with trypsin. Primary cultures were initiated in complete KGM2 semi-defined medium (Clonetics) on flasks coated with collagen type I (Falcon Biocoat). Sub-confluent cultures were re-plated to allow amplification through a second passage. Third passage cultures were seeded at 40 000 cells/cm2 and were grown at 37°C and 5% CO2. Cultures were confluent at day 5 and cells were further incubated for 3 days post-confluence in order to reach a morphological differentiated state.
Sham irradiated cells received the same treatment, except for irradiation. Cultures were then returned to the incubator and harvested at 3,6,15,24,48 and 72 hours following irradiation in order to prepare RNA extracts. Total RNA was amplified using the MessageAmp RNA Kit (Ambion). One round of amplification was performed. Equal amounts of amplified RNA obtained from mock irradiated cells at each time point (3,6,15,24,48 and 72 hours post-irradiation) were pooled together. Amplified RNA was labeled by coupling with aminoallyl-modified dUTP (Sigma), followed by a subsequent incorporation of monofunctionnal Cy3 or Cy5-N-succinimide esters (Amersham Biosciences). For each labeling experiment, amplified RNA (2 µg) was reverse-trancribed using random hexamers in the presence of 200 µM aminoallyl-dUTP, 500 µM each dATP, dCTP, dGTP, 300 µM dTTP, 400 U of Superscript II polymerase (Invitrogen) 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 H (Invitrogen) and incubating at 37°C for 15 minutes. The volume was adjusted to 400 µl with H20, aminoallyl-cDNA was purified by centrifugation in a microcon (YM-30 Millipore Amicon), eluted with 20 µl of water and then dried in a vacuum to 10µl. One µl sodium bicarbonate 0.5 M was added to aminoallyl-cDNA and subsequently mixed with vacuum-dried monofunctional dye. Coupling reaction was performed during 1 hour at room temperature in the dark. Labeled cDNAs were purified with a nucleospin column (Macherey Nagel), 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 1 volume of 5 M ammonium acetate in chilled ethanol.
The microarray slides were pre-hybridized in 3,5x SSC, 0,1% SDS and 1% BSA for 30 mn at 50°C. The labeled extract pellet was suspended in the hybridization solution (formamide 50%, 2,5x Denhardt, 0,5% SDS, 0,6% SSPE). The solution was heated to 95°C, added onto the prehybridized microarray slides, and hybridized at 42°C in a humidified chamber for 16 to 18 hours. Microarray glass slides were washed with 0,1X SSC / 0,1 % SDS at room temperature for 15 minutes and then twice with 0,1X SSC in the same conditions.

source_name: Differentiated primary keratinocyte culture, pool of equal quantities of amplified RNA obtained from sham irradiated cells at each time point (3h,6h,15h,24h,48h,72h).


title: CEA_SGF:AMD042_ASP


title: CEA_SGF:AMD040_ASP


title: CEA_SGF:ALE012_ASP


title: CEA_SGF:AMD028_ASP

source_name: Differentiated primary keratinocyte culture, 15 hours, 0 Gy


title: CEA_SGF:AMD027_ASP


title: CEA_SGF:AMD037_ASP


title: CEA_SGF:AMD032_ASP


title: CEA_SGF:AMD033_ASP


title: CEA_SGF:AMD031_ASP


title: CEA_SGF:ALY007_ASP


title: CEA_SGF:AMD030_ASP


title: CEA_SGF:AMD038_ASP


title: CEA_SGF:AMD039_ASP


title: CEA_SGF:ALY006_ASP


title: CEA_SGF:AMD025_ASP


title: CEA_SGF:AMD026_ASP


title: CEA_SGF:AMD034_ASP


title: CEA_SGF:AMD035_ASP


title: CEA_SGF:AMD029_ASP


title: CEA_SGF:AMD036_ASP


title: CEA_SGF:AMD024_ASP


title: CEA_SGF:ALY003_ASP


title: CEA_SGF:ALY005_ASP


title: CEA_SGF:ALY004_ASP


title: sugar, female 24, chip 3

city: Karlsruhe

first: Michael

middle: J.

last: Pankratz

department: Institute of Genetics

institute: Forschungszentrum Karlsruhe

description:
OregonR flies were kept in 50 ml PS-tubes flat bottom (Greiner) at 25¡C on standard fly food. For the control flies were moved to tubes containing yeast paste on PBS soaked filterpaper (normal), starved flies were obtained by putting them in tubes with PBS + 20% sucrose soaked filterpaper for 24h. For further details see publication.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = nutrient response, starvation, anti-aging, longevity
Lot batch = FP3_055

organism: Drosophila melanogaster

source_name: normal

source_name: 24h sugar


title: CEA_SGF:ALY011_ASP

source_name: Differentiated primary keratinocyte culture, 48 hours, 10 mGy


title: sugar, female 24, chip 5

description:
OregonR flies were kept in 50 ml PS-tubes flat bottom (Greiner) at 25¡C on standard fly food. For the control flies were moved to tubes containing yeast paste on PBS soaked filterpaper (normal), starved flies were obtained by putting them in tubes with PBS + 20% sucrose soaked filterpaper for 24h. For further details see publication.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = nutrient response, starvation, anti-aging, longevity
Lot batch = FP3_057


title: sugar, male 24h, chip 3

description:
OregonR flies were kept in 50 ml PS-tubes flat bottom (Greiner) at 25¡C on standard fly food. For the control flies were moved to tubes containing yeast paste on PBS soaked filterpaper (normal), starved flies were obtained by putting them in tubes with PBS + 20% sucrose soaked filterpaper for 24h. For further details see publication.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = nutrient response, starvation, anti-aging, longevity
Lot batch = FP3_054


title: sugar, male 48h, chip 3

description:
OregonR flies were kept in 50 ml PS-tubes flat bottom (Greiner) at 25¡C on standard fly food. For the control flies were moved to tubes containing yeast paste on PBS soaked filterpaper (normal), starved flies were obtained by putting them in tubes with PBS + 20% sucrose soaked filterpaper for 48h. For further details see publication.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = nutrient response, starvation, anti-aging, longevity
Lot batch = FP3_048

source_name: 48h sugar


title: sugar, male 24h, chip 6

description:
OregonR flies were kept in 50 ml PS-tubes flat bottom (Greiner) at 25¡C on standard fly food. For the control flies were moved to tubes containing yeast paste on PBS soaked filterpaper (normal), starved flies were obtained by putting them in tubes with PBS + 20% sucrose soaked filterpaper for 24h. For further details see publication.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = nutrient response, starvation, anti-aging, longevity
Lot batch = FP3_056


title: starvation, female 48h, chip 3

description:
OregonR flies were kept in 50 ml PS-tubes flat bottom (Greiner) at 25¡C on standard fly food. For the control flies were moved to tubes containing yeast paste on PBS soaked filterpaper (normal), starved flies were obtained by putting them in tubes with PBS soaked filterpaper only for 48h. For further details see publication.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = nutrient response, starvation, anti-aging, longevity
Lot batch = FP5_024

source_name: 48h starved


title: sugar, male 48h, chip 1

description:
OregonR flies were kept in 50 ml PS-tubes flat bottom (Greiner) at 25¡C on standard fly food. For the control flies were moved to tubes containing yeast paste on PBS soaked filterpaper (normal), starved flies were obtained by putting them in tubes with PBS + 20% sucrose soaked filterpaper for 48h. For further details see publication.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = nutrient response, starvation, anti-aging, longevity
Lot batch = FP3_046


title: sugar, male 48h, chip 4

description:
OregonR flies were kept in 50 ml PS-tubes flat bottom (Greiner) at 25¡C on standard fly food. For the control flies were moved to tubes containing yeast paste on PBS soaked filterpaper (normal), starved flies were obtained by putting them in tubes with PBS + 20% sucrose soaked filterpaper for 48h. For further details see publication.
The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link.
Keywords = nutrient response, starvation, anti-aging, longevity
Lot batch = FP5_025