title: Transdetermination study; prothoracic leg disc_AK93

description: These experiments are described in Klebes et al. (Development, 2005, in press). Comparisons – category #1: slide AK93, wildtype prothoracic leg disc – probe 1 vs. reference sample – probe2  (ratios are shown as probe2/probe1). Whole prothoracic leg imaginal discs of wildtype Drosophila larvae were compared to the common reference sample (control experiment). For this experiment leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. The common reference sample to which we compared control leg imaginal discs was generated by pooling amplified RNA of male and female 1st, 2nd and 3rd leg imaginal discs in equal proportions. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated. Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

tag: wildtype (OR) 1st leg disc

treatment_protocol: dissected discs, 2 rounds of linear amplification


title: Transdetermination study; wing vs leg_5-255

status: Public on Jul 08 2005

type: RNA

city: Berlin

first: Ansgar

last: Klebes

address: Chausseestr. 117

department: Biology

country: Germany

institute: Humboldt University Berlin

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #2,  slide 5-255, wing disc – probe 1 vs. mesothoracic leg disc – probe 2. (ratios are shown as probe2/probe1).  Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a mesothoracic (2nd) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

data_processing: log2 transformed global median normalized ratios (probe2/probe1)

molecule: total RNA

organism: Drosophila melanogaster

source_name: wing imaginal disc

treatment_protocol: dissected disc, 2 rounds of linear amplification

tag: wildtype (OR) wing imaginal disc of third instar male larva

source_name: mesothoracic leg imaginal disc

tag: wildtype (OR) mesothoracic leg imaginal disc of third instar male larva


title: Transdetermination study; prothoracic leg disc_5-085

description: These experiments are described in Klebes et al. (Development, 2005, in press). Comparisons – category #1: slide 5-131, reference sample – probe1 vs. wildtype prothoracic leg disc – probe 2 (ratios are shown as probe1/probe2). Whole prothoracic leg imaginal discs of wildtype Drosophila larvae were compared to the common reference sample (control experiment). For this experiment leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. The common reference sample to which we compared control leg imaginal discs was generated by pooling amplified RNA of male and female 1st, 2nd and 3rd leg imaginal discs in equal proportions. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated. Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

data_processing: log2 transformed global median normalized ratios (probe1/probe2)

source_name: prothoracic leg imaginal disc

source_name: leg disc reference sample

tag: pooled amplified RNA of 1st, 2nd and 3rd leg discs of third instar male and female wildtype larvae


title: Transdetermination study; prothoracic leg disc_5-131

description:
These experiments are described in Klebes et al. (Development, 2005, in press). Comparisons – category #1: slide 5-131, reference sample – probe1 vs. wildtype prothoracic leg disc – probe 2 (ratios are shown as probe1/probe2).
Whole prothoracic leg imaginal discs of wildtype Drosophila larvae were compared to the common reference sample (control experiment). For this experiment leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells. RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. The common reference sample to which we compared control leg imaginal discs was generated by pooling amplified RNA of male and female 1st, 2nd and 3rd leg imaginal discs in equal proportions. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated. Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Transdetermination study; wing vs leg_5-251

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #2,  slide 5-251, wing disc – probe 1 vs. prothoracic leg disc – probe 2. (ratios are shown as probe2/probe1).  Wing imaginal discs of single wildtype Drosophila larvae were compared to a single leg imaginal disc of the same animal. In this experiment the leg disc was a prothoracic (1st) leg imaginal disc. These comparisons were intended to establish the unique expression profiles of wing and leg imaginal discs. For this experiment wing and leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate wing and leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

tag: wildtype (OR) prothoracic leg imaginal disc of third instar male larva


title: Transdetermination study; prothoracic leg disc_5-069

description:
These experiments are described in Klebes et al. (Development, 2005, in press).

Comparisons – category #1:
slide 5-069, reference sample – probe1 vs. wildtype prothoracic leg disc – probe 2
(ratios are shown as probe1/probe2).

Whole prothoracic leg imaginal discs of wildtype Drosophila larvae were compared to the common reference sample (control experiment). For this experiment leg imaginal discs of third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate leg discs. Discs were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.

RNA was extracted from single imaginal discs using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.

The common reference sample to which we compared control leg imaginal discs was generated by pooling amplified RNA of male and female 1st, 2nd and 3rd leg imaginal discs in equal proportions.

Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.

Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.


title: Gene expression profiling of patient No. 18 (A)

status: Public on Jul 05 2005

city: Padova

first: Gerolamo

last: Lanfranchi

address: Via U. Bassi, 58/B

department: CRIBI - Biotechnology Center and Biology Department

country: Italy

institute: University of Padova

description:
An aliquot of 20-30 x106 leukemic bone marrow cells was used to prepare total RNA applying the Trizol (Invitrogen) purification method according to the manufacturers’ instructions.
Total RNA was retro-transcribed and labeled using a MICROMAX TSA Labeling Kit (PerkinElmer). Two ug of total RNA were used in each reaction but only half of the labeled cDNA was actually hybridized to the microarrays. Microarray hybridisation was carried out in a dual slide chamber (HybChamber, Gene Machines, San Carlos, CA, USA) humidified with 100 µl of 3 x SSC. Labeled cDNA was dissolved in 40 µl of hybridisation buffer, denatured at 90°C for 2 min in a thermal cycler and applied directly to the slides. Microarrays were covered with 22 x 40 mm cover slip and hybridized overnight at 65°C by immersion in a high precision water bath (W28, Grant, Cambridge, UK). Post-hybridization washing was performed according to the MICROMAX TSA Detection kit (PerkinElmer). . Digital images were generated in a GSI Lumonics LITE dual confocal laser scanner (ScanArray Microarray Analysis Software) and processed with QuantArray Analysis Software (GSI Lumonics, Ottawa, Canada). Normalized data were determined using the publicly available software MIDAS (TIGR Microarray Data Analysis System: http://www.tigr.org/softlab/). Normalized values (lowess normalization) were calculated for each spot, and converted in logarithmic scale. Final values correspond to log(2) ratio of the normalized intensities.
Keywords = human bone marrow
Keywords = childhood leukemia
Keywords = gene expression profiling

organism: Homo sapiens

source_name: normal human bone marrow (Reference)

source_name: leukemic bone marrow of patient No. 18 (Test)


title: Gene expression profiling of patient No. 17 (A)

source_name: leukemic bone marrow of patient No. 17 (Test)


title: Miniarray expression profiling of patient No. 34

description:
An aliquot of 20-30 x106 leukemic bone marrow cells was used to prepare total RNA applying the Trizol (Invitrogen) purification method according to the manufacturers’ instructions.
Total RNA was retro-transcribed and labeled using a MICROMAX TSA Labeling Kit (PerkinElmer). Two ug of total RNA were used in each reaction but only half of the labeled cDNA was actually hybridized to the microarrays. Microarray hybridisation was carried out in a dual slide chamber (HybChamber, Gene Machines, San Carlos, CA, USA) humidified with 100 µl of 3 x SSC. Labeled cDNA was dissolved in 40 µl of hybridisation buffer, denatured at 90°C for 2 min in a thermal cycler and applied directly to the slides. Microarrays were covered with 22 x 40 mm cover slip and hybridized overnight at 65°C by immersion in a high precision water bath (W28, Grant, Cambridge, UK). Post-hybridization washing was performed according to the MICROMAX TSA Detection kit (PerkinElmer). . Digital images were generated in a GSI Lumonics LITE dual confocal laser scanner (ScanArray Microarray Analysis Software) and processed with QuantArray Analysis Software (GSI Lumonics, Ottawa, Canada). Normalized data were determined using the publicly available software MIDAS (TIGR Microarray Data Analysis System: http://www.tigr.org/softlab/). Normalized values (global mean normalization) were calculated for each spot, and converted in logarithmic scale. Final values correspond to log(2) ratio of the normalized intensities
Keywords = human bone marrow
Keywords = childhood leukemia
Keywords = gene expression profiling
Keywords = miniarray

source_name: leukemic bone marrow of patient No. 34 (Test)


title: Miniarray expression profiling of patient No. 35

source_name: leukemic bone marrow of patient No. 35 (Test)


title: Gene expression profiling of patient No. 17 (B)


title: Gene expression profiling of patient No. 14 (B)

source_name: leukemic bone marrow of patient No. 14 (Test)


title: Gene expression profiling of patient No. 15 (B)

source_name: leukemic bone marrow of patient No. 15 (Test)


title: Gene expression profiling of patient No. 11 (B)

source_name: leukemic bone marrow of patient No. 11 (Test)


title: Gene expression profiling of patient No. 11 (A)


title: Gene expression profiling of patient No. 13 (B)

source_name: leukemic bone marrow of patient No. 13 (Test)


title: Gene expression profiling of patient No. 14 (A)


title: Gene expression profiling of patient No. 12 (A)

source_name: leukemic bone marrow of patient No. 12 (Test)


title: Transdetermination study; TD cells vs V(wg) cells_7-204

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #5,  slide 7-204, V(wg) cells – probe 1 vs. TD cells– probe 2 (ratios are represented as probe1/probe2).  Unique cell populations of Wingless over-expressing prothoracic leg imaginal discs of y,w,hs-flp; vgBE-GAL4/UASGFP, Actinc5, FRT y+, FRT, wg+ Drosophila larvae were compared with each other. In this experiment, cells that undergo transdetermination (TD cells) were compared to ventral cells (V(wg)) isolated from the same discs. These comparisons were intended to establish the unique expression profiles of leg imaginal cells while they undergo a fate switch to transdetermine into wing cells.  Prothoracic leg imaginal disc cells of the “weak point” that are competent to undergo transdetermination (TD cells) were labeled in vivo using the vgBE::GAL4/UAS-GFP reporter system (Sustar and Schubiger, 2005). Transdetermination was induced in these discs by over-expressing Wingless. TD cells were micro-dissected under a fluorescent dissecting scope. The remaining cells of the leg disc were separated into a dorsal (D(wg)) that lack the TD cells and ventral (V(wg)) fraction by cutting approximately along the median of the disc. Pooled fragments from 4-8 discs were used  Ectopic Wg expression was induced by heat shock of young L3 larvae 60 h and 72h after egg deposition, and leg discs were isolated three days later. This protocol results in Wg over-expression in over 90% of the disc cells after heat shock (Maves and Schubiger, 1995). Note that Wg over-expression delays metamorphosis by one day, so these larvae had not yet pupariated. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: Transdetermining (TD) cells

tag: microdissected TD cells from Wingless over-expressing prothoracic leg imaginal discs, 2 rounds of linear amplification

source_name: Ventral (V(wg)) cells

tag: microdissected ventral cells from Wingless over-expressing prothoracic leg imaginal discs (V(wg)), 2 rounds of linear amplification


title: Gene expression profiling of patient No. 10 (B)

source_name: leukemic bone marrow of patient No. 10 (Test)


title: Gene expression profiling of patient No. 16 (B)

source_name: leukemic bone marrow of patient No. 16 (Test)


title: Gene expression profiling of patient No. 15 (A)


title: Gene expression profiling of patient No. 16 (A)


title: Gene expression profiling of patient No. 9 (B)

source_name: leukemic bone marrow of patient No. 9 (Test)


title: Gene expression profiling of patient No. 9 (A)


title: Gene expression profiling of patient No. 13 (A)


title: Gene expression profiling of patient No. 12 (B)


title: Gene expression profiling of patient No. 10 (A)


title: Gene expression profiling of patient No. 18 (B)


title: Gene expression profiling of patient No. 8 (B)

source_name: leukemic bone marrow of patient No. 8 (Test)


title: Miniarray expression profiling of patient No. 33

source_name: leukemic bone marrow of patient No. 33 (Test)


title: Miniarray expression profiling of patient No. 32

source_name: normal human bone marrow (Reference)

source_name: leukemic bone marrow of patient No. 32 (Test)


title: Gene expression profiling of patient No. 8 (A)


title: Gene expression profiling of patient No. 7 (B)

source_name: leukemic bone marrow of patient No. 7 (Test)


title: Miniarray expression profiling of patient No. 30

source_name: leukemic bone marrow of patient No. 30 (Test)


title: Miniarray expression profiling of patient No. 31

source_name: leukemic bone marrow of patient No. 31 (Test)


title: Gene expression profiling of patient No. 7 (A)


title: Gene expression profiling of patient No. 5 (B)

source_name: leukemic bone marrow of patient No. 5 (Test)


title: Gene expression profiling of patient No. 6 (A)

source_name: leukemic bone marrow of patient No. 6 (Test)


title: Gene expression profiling of patient No. 6 (B)


title: Miniarray expression profiling of patient No. 27

source_name: leukemic bone marrow of patient No. 27 (Test)


title: Miniarray expression profiling of patient No. 25

source_name: leukemic bone marrow of patient No. 25 (Test)


title: Miniarray expression profiling of patient No. 24

source_name: leukemic bone marrow of patient No. 24 (Test)


title: Miniarray expression profiling of patient No. 23

source_name: leukemic bone marrow of patient No. 23 (Test)


title: Miniarray expression profiling of patient No. 26

source_name: leukemic bone marrow of patient No. 26 (Test)


title: Miniarray expression profiling of patient No. 29

source_name: leukemic bone marrow of patient No. 29 (Test)


title: Miniarray expression profiling of patient No. 22

source_name: leukemic bone marrow of patient No. 22 (Test)


title: Miniarray expression profiling of patient No. 21

source_name: leukemic bone marrow of patient No. 21 (Test)


title: Miniarray expression profiling of patient No. 20

source_name: leukemic bone marrow of patient No. 20 (Test)


title: Miniarray expression profiling of patient No. 28

source_name: leukemic bone marrow of patient No. 28 (Test)


title: Gene expression profiling of patient No. 5 (A)

source_name: normal human bone marrow (Reference)


title: Gene expression profiling of patient No. 3 (B)

source_name: leukemic bone marrow of patient No. 3 (Test)


title: Miniarray expression profiling of patient No. 19

source_name: leukemic bone marrow of patient No. 19 (Test)


title: Gene expression profiling of patient No. 4 (B)

source_name: leukemic bone marrow of patient No. 4 (Test)


title: MM 1B

status: Public on Jul 01 2005

first: Kai

middle: Martin

last: Schmidt-Ott

address: Robert-Roessle-Str. 10

department: Differentiation and Regeneration of Kidney Epithelia

institute: Max-Delbrueck Center for Molecular Medicine

description:
Metanephric mesenchyme and ureteric buds were dissected from E13.5  rats.  At this stage of development, the ureteric buds have already initiated branching morphogenesis producing cleavage of the initial bulbous ureteric bud into a T-shaped tubule. To isolate the metanephric mesenchyme and ureteric bud, embryonic kidneys were dissected and treated with DNase (100 U/ml; Boehringer Mannheim) and trypsin (1 mg/ml; Sigma) for 10 min at 37C in L-15 media. The mesenchyme was freed with minutia pins (Fine Scientific) and the tissues were transferred to L-15 with soybean trypsin inhibitor (1 mg/ml). Cross-contamination between ureteric buds and metanephric mesenchymes was ruled out by visual inspection and staining with dolichos bifloris lectin, which selectively stains the ureteric bud at this stage. In all, approximately 1,200 rat ureteric buds and 240 rat mesenchymes were harvested to prepare RNA. Total RNA from ureteric buds and metanephric mesenchymes was extracted using RNeasy mini kit (Qiagen, Valencia, CA) with on-column DNase digestion according to the manufacturer´s instructions. Targets were produced using standard Affymetrix procedures from 1 ug of total RNA.  2 replicates of the target labeling process were analyzed for each sample.
Keywords = Rattus norvegicus, metanephric mesenchyme, ureteric bud, molecular screen for spatially restricted transcripts

organism: Rattus norvegicus

source_name: E13.5 metanephric mesenchyme


title: Gene expression profiling of patient No. 3 (A)

description:
An aliquot of 20-30 x106 leukemic bone marrow cells was used to prepare total RNA applying the Trizol (Invitrogen) purification method according to the manufacturers’ instructions.
Total RNA was retro-transcribed and labeled using a MICROMAX TSA Labeling Kit (PerkinElmer). Two ug of total RNA were used in each reaction but only half of the labeled cDNA was actually hybridized to the microarrays. Microarray hybridisation was carried out in a dual slide chamber (HybChamber, Gene Machines, San Carlos, CA, USA) humidified with 100 µl of 3 x SSC. Labeled cDNA was dissolved in 40 µl of hybridisation buffer, denatured at 90°C for 2 min in a thermal cycler and applied directly to the slides. Microarrays were covered with 22 x 40 mm cover slip and hybridized overnight at 65°C by immersion in a high precision water bath (W28, Grant, Cambridge, UK). Post-hybridization washing was performed according to the MICROMAX TSA Detection kit (PerkinElmer). . Digital images were generated in a GSI Lumonics LITE dual confocal laser scanner (ScanArray Microarray Analysis Software) and processed with QuantArray Analysis Software (GSI Lumonics, Ottawa, Canada). Normalized data were determined using the publicly available software MIDAS (TIGR Microarray Data Analysis System: http://www.tigr.org/softlab/). Normalized values (lowess normalization) were calculated for each spot, and converted in logarithmic scale. Final values correspond to log(2) ratio of the normalized intensities.

Keywords = human bone marrow
Keywords = childhood leukemia
Keywords = gene expression profiling


title: MM 2B


title: Gene expression profiling of patient No. 4 (A)


title: Gene expression profiling of patient No. 2 (B)

source_name: leukemic bone marrow of patient No. 2 (Test)


title: UB 1B

source_name: E13.5 ureteric bud


title: UB 2B


title: UB 1C


title: MM 2C


title: MM 1


title: UB 2C


title: MM 1C


title: UB 2


title: MM 2


title: UB 1


title: 100% adult mouse colon

status: Public on Jun 30 2005

city: Cincinnati

first: Johannes

middle: Michael

last: Freudenberg

state: OH

address: 3333 Burnet Ave

department: Biomedical Informatics

country: USA

institute: Cincinnati Children's Hospital Medical Center

description: 8 week old adult C57BL/6 mouse colons. homogenized tissue in Trizol reagent (Invitrogen) with a Polytron tissue homogenizer (Brinkman, Westbury, NY)).  Prepared as a pooled single sample from multiple animals.  Purified RNA following the manufacturer’s recommendations and integrity was analyzed using an Agilent BioAnalyzer 2100 according to manufacturer instructions.

hyb_protocol: according to manufacturer's instructions

data_processing: RMA

organism: Mus musculus

source_name: 8 week old adult C57BL/6 mouse colon

label_protocol: T7 double amplification

extract_protocol: from 10ng of total RNA

tag: Strain

tag: Tissue

value: colon

tag: Sample

value: pooled


title: 50% whole mouse 50% mouse colon

description: Two reference RNA samples were prepared, one from 8 week old adult C57BL/6 mouse colons and one from one day old C57BL/6 whole pups by homogenizing tissue in Trizol reagent (Invitrogen) with a Polytron tissue homogenizer (Brinkman, Westbury, NY).  Both samples were prepared as a pooled single sample from multiple animals.  RNA was purified following the manufacturer’s recommendations and integrity was analyzed using an Agilent BioAnalyzer 2100 according to manufacturer instructions.  A dilution series of five sample RNAs was prepared from the two reference RNAs by mixing the one day old whole mouse RNA and the 8-week old colon RNA.

hyb_protocol: according to Affymetrix' recommendations

source_name: C57BL/6 whole mouse/mouse colons

label_protocol: T7 double amplification protocol (Schwab et al., 2003)

value: C57BL/6 Tissue:whole mouse/mouse colon Sample:pooled


title: M JH 16 h 140298

status: Public on Jun 16 2005

city: Reno

first: Christopher

middle: Ian

last: Keeling

state: NV

address: 1664 North Virginia Street

department: Department of Biochemistry/Mail Stop 330

institute: University of Nevada, Reno

description:
Tissue: Logging slash of Jeffrey pine (Pinus jeffreyi Grev. & Balf.) infested with Ips pini were obtained from Ward Creek CA (39*08'41" N, 120*12'01" W, 2048 m) and placed in a greenhouse to allow emergence of adult beetles (Browne, J. Econ. Entomol. 65, 1499-1501, 1972). Upon emergence, beetles were typed and sexed and kept at 4°C on moist paper towels. Beetles were immobilized by chilling and then treated topically on the ventral surface of the abdomen with either 10 µg (±) juvenile hormone III (Sigma) in 0.5 µL acetone or acetone (control). They were then incubated in the dark at room temperature in groups of 10 in 60 mL plastic containers (Dixie) for specified times.  At the end of the incubation, beetles were immersed in water and the anterior midguts excised under a stereo microscope. Midguts were gently purged of their contents and then immediately frozen in liquid nitrogen and stored at -80°C. Each treatment/control time point pair was replicated four times and each replicate contained tissue from 60 midguts (30 each for treatment and control). Treatment and control beetles were cohorts for a replicate pair and were incubated simultaneously. Beetles in different replicate pairs and time points were not always cohorts.
Hybridizations: Hybridizations were done in quadruplicate, each with tissue from different groups of beetles. Total RNA was extracted using QIAshredder and RNeasy Mini kits (Qiagen) and quantified spectrophotometrically. Total RNA (approx. 40 µg) was reverse transcribed with oligo(dT)15 (Promega), Superscript III RT- (Invitrogen), and dNTPs (Promega) containing aminoallyl-dUTP (Sigma, aminoallyl-dUTP to dTTP ratio of 2 to 3). After alkaline hydrolysis of the RNA, clean up with a Microcon-30 (Millipore), and lyophilization, the aminoallyl-dUTP cDNA was reacted with Cy3 or Cy5 monofunctional reactive dyes (Amersham). Cy3 and Cy5 dyes were swapped equally between treatment and control samples among replicates (see source info for dye used for each target). After 60 min, unreacted dyes were removed from the labeled cDNA by a QIAquick spin column (Qiagen). The fluorescently labeled cDNAs from treatment and control samples were combined together with 20 µg herring sperm DNA (Promega) and 20 µg polydA (Amersham), lyophilized, and then resuspended in 20 µL hybridization buffer (50% formamide, 5x SSC, and 0.1% SDS). The hybridization mixture was denatured at 95°C for 5 min, centrifuged at 16,000 g for 5 min, and then immediately applied to the microarray preheated to 42°C. Prior to hybridization, microarrays were washed 5 min in 0.1% SDS, 5 min in 2x SSC, denatured 3 min in boiling water, placed into ice-cold ethanol, dried by centrifugation, incubated for 60 min at 42°C in pre-hybridization buffer (5x SSC, 0.1% SDS, and 1% BSA), washed 5 min in water, 5 min in 2-propanol, and then dried by centrifugation. The hybridization mixture was covered with a HybriSlip (22 x 40 mm, Sigma) and then the microarray was immediately placed into a preheated, humidified hybridization chamber and placed in a 42°C oven for 16-20 h. Microarrays were removed from the hybridization chamber and immediately washed with agitation at room temperature for 5 min each in 2x SSC with 0.03% SDS, 1x SSC, and 0.2x SSC, and then dried by centrifugation.
Analysis: Arrays were scanned at 5 µm resolution with a Packard Bioscience ScanArray 4000 array scanner and analyzed with ScanArray and QuantArray software (versions 3.1 and 3.0 respectively). Median signal (adaptive spot algorithm) and background intensities were obtained for both dyes for each element. Data were background subtracted, globally and locally (across microarray surface, span=0.5, trim=0.1) normalized, log transformed (base 2), converted to mean log (intensities) and log (ratios), and then locally normalized across element signal intensity (span=0.7, trim=0.1) using online SNOMAD tools (Colantuoni et al., Bioinformatics 18, 1540-1, 2002). "Empty" and "Spotting Soln" elements were excluded from analysis by SNOMAD.  Data for these two element types were arbitrarily filled with zeros to upload into NCBI GEO.  To account for outliers, the elements in triplicate on the 4 replicated arrays were treated as unique measurements and a Q-test (96% confidence, Dean and Dixon, Anal. Chem. 23, 636-8, 1951) was performed on the 12 measurements. If a measurement tested as an outlier, its VALUE was replaced with the average VALUE of the two other replicate elements on the same array.  At most, only 1 of the 12 measurements was excluded.
Keywords = North American pine engraver beetle, anterior midgut, juvenile hormone, pheromone biosynthesis, Coleoptera, Scolytidae

organism: Ips pini

source_name: Anterior midguts from acetone treated males, 16 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated males, 16 hours (Cy5)


title: 25% whole mouse 75% mouse colon

scan_protocol: according to Affymetrix' recommendations


title: 100% whole mouse

description:
one day old C57BL/6 whole mouse.
homogenized tissue in Trizol reagent (Invitrogen) with a Polytron tissue homogenizer (Brinkman, Westbury, NY). pooled single sample from multiple animals
RNA purified following the manufacturer’s recommendations.

scan_protocol: according to manufacturer's instructions

source_name: one day old C57BL/6 whole mouse pooled RNA

value: whole mouse


title: M JH 8 h 140294

source_name: Anterior midguts from acetone treated males, 8 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated males, 8 hours (Cy3)


title: M JH 16 h 157324

source_name: Anterior midguts from acetone treated males, 16 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated males, 16 hours (Cy3)


title: M JH 16 h 140295


title: M JH 8 h 150850


title: M JH 16 h 157323


title: M JH 8 h 150849

source_name: Anterior midguts from acetone treated males, 8 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated males, 8 hours (Cy5)


title: M JH 4 h 150834

source_name: Anterior midguts from acetone treated males, 4 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated males, 4 hours (Cy5)


title: M JH 4 h 150839


title: M JH 8 h 140293


title: 75% whole mouse 25% mouse colon

label: according to Affymetrix' recommendations

value: whole mouse/mouse colon


title: M JH 4 h 150835

source_name: Anterior midguts from acetone treated males, 4 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated males, 4 hours (Cy3)


title: M JH 2 h 150831

source_name: Anterior midguts from acetone treated males, 2 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated males, 2 hours (Cy3)


title: F JH 16 h 157322

source_name: Anterior midguts from acetone treated females, 16 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated females, 16 hours (Cy5)


title: M JH 2 h 150826

source_name: Anterior midguts from acetone treated males, 2 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated males, 2 hours (Cy5)


title: M JH 2 h 150827


title: F JH 16 h 140309

source_name: Anterior midguts from acetone treated females, 16 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated females, 16 hours (Cy3)


title: F JH 16 h 157321


title: M JH 2 h 150830


title: F JH 16 h 140302


title: F JH 8 h 140292

source_name: Anterior midguts from acetone treated females, 8 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated females, 8 hours (Cy3)


title: F JH 4 h 150841

source_name: Anterior midguts from acetone treated females, 4 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated females, 4 hours (Cy5)


title: F JH 4 h 150842

source_name: Anterior midguts from acetone treated females, 4 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated females, 4 hours (Cy3)


title: F JH 8 h 150848


title: MPM cell line MS589

status: Public on Jun 01 2005

city: Boston

first: Gavin

last: Gordon

state: MA

institute: Brigham and Women's Hospital/Harvard Medical School

description: Discarded MPM surgical specimens (n=40), normal pleura specimens (n=5), and normal lung specimens (n=4) were freshly collected (and snap frozen) from patients who underwent surgery at Boston’s Brigham and Women’s Hospital (BWH) between October 1998 and August 2000.  All of these patients underwent extrapleural pneumonectomy with heated intra-pleural cisplatin chemotherapy delivered after the specimens were removed.  All normal specimens were obtained from patients who were never diagnosed with MPM.  Two human MPM cell lines (MS589 and MS428) were kindly provided by Jonathan A. Fletcher, M.D., Department of Pathology, BWH.  The JMN1B MPM cell line19,20 has been described previously.  The SV40-immortalized, non-tumorigenic mesothelial cell line (Met-5A)21 and the MPM cell line MSTO-211H22 were purchased from the American Type Culture Collection.  Normal tissues were obtained from additional consented patients undergoing treatment for diseases other than MPM.  All MPM samples used in these studies contained relatively pure tumor (greater than 50% tumor cells per high power field examined in a section adjacent to the tissue used).  The microscopic slides from the patients’ resection specimens were reviewed by one of the authors (J.G.), and the diagnosis and histologic subclassification of MPM confirmed in all cases.  Linked clinical and pathological data were obtained for all patients who contributed tumor specimens.  Specimens and data were rendered anonymous to protect patient confidentiality.  Studies utilizing human tissues were approved by and conducted in accordance with the policies of the Institutional Review Board at BWH.

source_name: MPM cell line MS589


title: F JH 2 h 150828

source_name: Anterior midguts from acetone treated females, 2 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated females, 2 hours (Cy5)


title: MPM cell line MS428

source_name: MPM cell line MS428


title: F JH 2 h 150832


title: F JH 8 h 150847

source_name: Anterior midguts from acetone treated females, 8 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated females, 8 hours (Cy5)


title: MPM cell line MSTO-211H

source_name: MPM cell line MSTO-211H


title: F JH 2 h 150833

source_name: Anterior midguts from acetone treated females, 2 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated females, 2 hours (Cy3)


title: MPM cell line JMN1B

source_name: MPM cell line JMN1B


title: F JH 8 h 140291


title: Non-tumorigenic mesothelial cell line Met-5A

source_name: non-tumorigenic mesothelial cell line Met-5A


title: Normal Lung 4

source_name: Normal Lung 4


title: Normal Pleura 4

source_name: Normal Pleura 4


title: Normal Lung 1

source_name: Normal Lung 1


title: Normal Pleura 3

source_name: Normal Pleura 3


title: F JH 4 h 150838

source_name: Anterior midguts from acetone treated females, 4 hours (Cy5)


title: Normal Lung 3

source_name: Normal Lung 3


title: F JH 2 h 150829


title: MPM tumor 42

source_name: MPM tumor specimen 42


title: MPM tumor 40

source_name: MPM tumor specimen 40


title: Normal Pleura 2

source_name: Normal Pleura 2


title: Normal Pleura 1

source_name: Normal Pleura 1


title: MPM tumor 39

source_name: MPM tumor specimen 39


title: M JH 4 h 150840

source_name: Anterior midguts from acetone treated males, 4 hours (Cy5)

source_name: Anterior midguts from juvenile hormone III treated males, 4 hours (Cy3)


title: MPM tumor 41

source_name: MPM tumor specimen 41


title: MPM tumor 33

source_name: MPM tumor specimen 33


title: MPM tumor 29

source_name: MPM tumor specimen 29


title: MPM tumor 36

source_name: MPM tumor specimen 36


title: MPM tumor 28

source_name: MPM tumor specimen 28


title: MPM tumor 30

source_name: MPM tumor specimen 30


title: MPM tumor 38

source_name: MPM tumor specimen 38


title: MPM tumor 37

source_name: MPM tumor specimen 37


title: MPM tumor 32

source_name: MPM tumor specimen 32


title: MPM tumor 31

source_name: MPM tumor specimen 31


title: MPM tumor 25

source_name: MPM tumor specimen 25


title: MPM tumor 27

source_name: MPM tumor specimen 27


title: MPM tumor 34

source_name: MPM tumor specimen 34


title: MPM tumor 26

source_name: MPM tumor specimen 26


title: MPM tumor 20

source_name: MPM tumor specimen 20


title: MPM tumor 23

source_name: MPM tumor specimen 23


title: MPM tumor 21

source_name: MPM tumor specimen 21


title: MPM tumor 17

source_name: MPM tumor specimen 17


title: MPM tumor 19

source_name: MPM tumor specimen 19


title: MPM tumor 18

source_name: MPM tumor specimen 18


title: MPM tumor 15

source_name: MPM tumor specimen 15


title: MPM tumor 16

source_name: MPM tumor specimen 16


title: MPM tumor 22

source_name: MPM tumor specimen 22


title: MPM tumor 14

source_name: MPM tumor specimen 14


title: MPM tumor 12

source_name: MPM tumor specimen 12


title: MPM tumor 13

source_name: MPM tumor specimen 13


title: MPM tumor 10

source_name: MPM tumor specimen 10


title: MPM tumor 5

source_name: MPM tumor specimen 5


title: MPM tumor 4

source_name: MPM tumor specimen 4


title: MPM tumor 8

source_name: MPM tumor specimen 8


title: MPM tumor 11

source_name: MPM tumor specimen 11


title: Differentiated Omental Replicate 1

city: Cambridge

first: Garrett

middle: M

last: Frampton

address: 9 Cambridge Center

institute: Whitehead Instute - MIT

description:
Trizol isolation of total RNA from Differentiated Human Omental Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.9, Fasting Glucose 114 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 3.037, Corner + 98, Corner -  98, Background 78.59, Noise 4.36, Raw Q 3.07
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Telomerase Expressing Clone Omental Replicate 2

description:
Trizol isolation of total RNA from Telomerase Expressing Clone of Human Omental Preadipocytes.
Subject: Caucasian Female, 42 YO, BMI 60.3, Fasting Glucose 87 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 2.903, Corner + 64, Corner -  64, Background 46.96, Noise 2.71, Raw Q 1.92
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Telomerase Expressing Clone Subcutaneous Replicate 3

description:
Trizol isolation of total RNA from Telomerase Expressing Clone of Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 42 YO, BMI 60.3, Fasting Glucose 87 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 2.531, Corner + 67, Corner -  67, Background 52.75, Noise 3.18, Raw Q 2.10
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: MPM tumor 2

source_name: MPM tumor specimen 2


title: MPM tumor 3

source_name: MPM tumor specimen 3


title: Telomerase Expressing Clone Omental Replicate 1

description:
Trizol isolation of total RNA from Telomerase Expressing Clone of Human Omental Preadipocytes.
Subject: Caucasian Female, 42 YO, BMI 60.3, Fasting Glucose 87 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 2.344, Corner + 70, Corner -  70, Background 45.66, Noise 2.90, Raw Q 1.87
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Telomerase Expressing Clone Subcutaneous Replicate 2

description:
Trizol isolation of total RNA from Telomerase Expressing Clone of Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 42 YO, BMI 60.3, Fasting Glucose 87 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 3.581, Corner + 61, Corner -  61, Background 37.28, Noise 2.02, Raw Q 1.56
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Omental Replicate 3

description:
Trizol isolation of total RNA from Differentiated Human Omental Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.4, Fasting Glucose 99 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 3.116, Corner + 72, Corner -  72, Background 72.81, Noise 3.98, Raw Q 2.96
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: MPM tumor 1

source_name: MPM tumor specimen 1


title: Telomerase Expressing Clone Omental Replicate 3

description:
Trizol isolation of total RNA from Telomerase Expressing Clone of Human Omental Preadipocytes.
Subject: Caucasian Female, 42 YO, BMI 60.3, Fasting Glucose 87 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 2.667, Corner + 67, Corner -  67, Background 42.86, Noise 2.55, Raw Q 1.75
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Telomerase Expressing Clone Subcutaneous Replicate 1

description:
Trizol isolation of total RNA from Telomerase Expressing Clone of Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 42 YO, BMI 60.3, Fasting Glucose 87 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 2.043, Corner + 86, Corner -  86, Background 40.61, Noise 2.90, Raw Q 1.79
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Mesenteric Replicate 3

description:
Trizol isolation of total RNA from Differentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.4, Fasting Glucose 99 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 2.335, Corner + 98, Corner -  98, Background 76.19, Noise 4.24, Raw Q 3.08
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Mesenteric Replicate 2

description:
Trizol isolation of total RNA from Differentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.1, Fasting Glucose 102 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 2.087, Corner + 94, Corner -  94, Background 60.09, Noise 3.19, Raw Q 2.51
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: MPM tumor 9

source_name: MPM tumor specimen 9


title: MPM tumor 6

source_name: MPM tumor specimen 6


title: Differentiated Omental Replicate 2

description:
Trizol isolation of total RNA from Differentiated Human Omental Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.1, Fasting Glucose 102 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 3.014, Corner + 78, Corner -  78, Background 60.44, Noise 3.14, Raw Q 2.51
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: MPM tumor 7

source_name: MPM tumor specimen 7


title: Undifferentiated Omental Replicate 2

description:
Trizol isolation of total RNA from Undifferentiated Human Omental Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.4, Fasting Glucose 99 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 4.599, Corner + 55, Corner -  55, Background 62.63, Noise 2.57, Raw Q 2.43
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Mesenteric Replicate 1

description:
Trizol isolation of total RNA from Differentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.9, Fasting Glucose 114 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 1.944, Corner + 88, Corner -  88, Background 70.77, Noise 3.45, Raw Q 2.72
Keywords = adipocyte, subcutaneous, mesenteric, omental